Anti-cholesterolemic compounds and methods of use

ABSTRACT

The present invention provides novel compounds with hypocholesteremic activity from crude  Embilica officinialis  (EO) extracts and methods of use. The invention also provides nutraceuticals.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/946,558, filed Jul. 19, 2013, which is a continuation of U.S.application Ser. No. 12/488,965, filed Jun. 22, 2009 (now U.S. Pat. No.8,492,351), which is a continuation of International Application No.PCT/US2007/088780 (WO 2008/080162), filed Dec. 24, 2007, which claimsthe benefit of U.S. Provisional Application 60/876,761, filed Dec. 22,2006, U.S. Provisional Application 60/877,753, filed Dec. 29, 2006, U.S.Provisional Application 60/876,599, filed Dec. 22, 2006, and U.S.Provisional Application 60/877,740, filed Dec. 29, 2006. The entirecontents of each of the aforementioned applications are herebyincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant nos.DK-31771 and DK-31722. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention provides novel compounds with hypocholesteremicactivity from Embilica offcinalis (EO) extracts and methods of use. Theinvention provides nutraceuticals. The invention relates to thedetermination of the biological activity and mechanism of action bywhich the purified compounds lower cholesterol levels.

BACKGROUND OF THE INVENTION

According to the American Heart Association, an estimated 100,870,000American adults have total cholesterol levels in the borderline-highrisk range of 200 mg/dl to 239 mg/dl, and there are 40,600,000 Americanadults living with high-risk cholesterol levels of 240 mg/dl or more.There are many risk factors that can indicate a propensity to have highlevels of cholesterol, such as age, weight, and health conditions suchas diabetes, smoking, gender, race and ethnicity.

Hypercholesterolemia, or elevated blood cholesterol levels due toconcentration of cholesterol in the cells and plasma, is an importantrisk factor definitively connected with potentially deadlycardiovascular disease, including atherosclerosis, coronary arteryinsufficiency, coronary heart disease, myocardial infarction and stroke.Millions of people around the world suffer from coronary heart disease,and it is the leading cause of death and morbidity at a productive age,especially in Western Europe and in the United States. Accordingly,cardiovascular disease presents a significant drain on healthcareresources in the western world. In the United States, total costs(direct and indirect) connected with the disease were estimated as about$118 billion in 2000; for 1 1 million citizens that experiencedmyocardial infarction, more than 40% of those died [Terry A. Jacobson,Clinical Context: Current Concepts of Coronary Heart Disease Management,Am J Med. 2001; 110 (6A):3S-11S]. In addition, cardiovascular disease isgrowing at an alarming rate in Asian countries, and in particular amongAsian Indians where cardiovascular disease has reached epidemicproportions W.

Cholesterol (and its derivatives) biosynthesis underlies cardiovasculardisease and, accordingly, inhibitors of cholesterol biosynthesis are amajor focus of research efforts towards new therapeutics forcardiovascular disease. For a number of years, significant research wasaimed at the development of competitive inhibitors for3-hydroxy-3-methylglutaryl coenzyme A reductase, a major regulatoryenzyme of cholesterol biosynthesis. Many attempts to use for thispurpose oxygenated sterols, which via binding oxysterol receptors wereexpected to decrease activity of HMG-CoA reductase, did not bringpractical results. A series of fungal metabolites with very highaffinities for HMG-CoA reductase were found to be highly efficientinhibitors of cholesterol biosynthesis. These compounds and somesynthetic analogs are commonly known as statins, are availablecommercially, and are widely used.

Although being relatively safe and efficient in treatment and preventionof coronary heart disease, statins have certain limitations in their usebecause of possible deleterious side effects, such as muscle weakness,and renal failure. Statin therapy is contra-indicated in pregnant womenand patients with liver disorders. Additionally, there is a significantpatient population in whom statins are not effective, and so the needfor an agent that will lower cholesterol extends beyond the number ofpatients currently taking statins to lower their cholesterol levels.Taking additionally into account the relatively high costs of statintherapy, which varies from $20,000 to $40,000 per quality-adjustedlife-year saved [John A. Farmer, Economic Implications of Lipid-LoweringTrials: Current Considerations in Selecting a Statin, Am J Cardiol 1998;82:26M-31M], and the desirability of long-term permanent treatment[Terry A. Jacobson, Clinical Context: Current Concepts of Coronary HeartDisease Management, Am J Med. 2001; 110 (6A):3S-11S], it is evident thatnew agents are needed with similar targeting as the statins but withhigher potency, safety, and availability and that will providesignificant cost savings.

Accordingly, the instant invention provides novel compounds withanticholesterolemic activity, and methods of use.

SUMMARY

The present invention relates generally to methods and compositions forreducing hypercholesteremia in a subject. The invention is based on theidentification of novel compounds from crude Embilica officinialis (EO)extracts. The invention relates to the determination of the biologicalactivity and mechanism of action by which the purified compounds lowercholesterol levels. The invention provides nutraceuticals.

In a first aspect, the invention provides a method for preventing ortreating an elevated blood lipid level-related disease or disorder in asubject comprising administering to the subject an effective amount ofone or more gallic acid derivatives, thereby preventing or treating anelevated blood lipid level-related disease or disorder in the subject.

In another aspect, the invention provides a method for preventing ortreating inflammation in a subject comprising administering to thesubject an effective amount of one or more gallic acid derivatives,thereby preventing or treating inflammation in the subject.

In still another aspect, the invention provides a method for preventingor treating a stress response in a subject comprising administering tothe subject an effective amount of one or more gallic acid derivatives,thereby preventing or treating a stress response in the subject.

In one embodiment of any one of the above aspects, the one or moregallic acid derivatives is administered as a nutraceutical.

In one aspect, the present invention provides a nutraceutical comprisingone or more gallic acid derivatives.

In a further aspect, the present invention provides a nutraceuticalcomprising one or more extracts from EuMil.

In another embodiment of any one of the above aspects, the one or moregallic acid derivatives are selected from the group consisting of:methyl gallate, ethyl gallate, glycerol-1-gallate, glucose-1-gallate(GG1), glucose-6-gallate (GG6), glucose-1,6-digallate (DGG16), mucicacid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone5-gallate, geraniin, corilagin, chebilc acid, and m-digallic acid withminor p-digallic acid.

In another particular embodiment, the one or more gallic acidderivatives are selected from the group consisting of: Compound4+Compound 5+Compound 2a, Compound 4+Compound 8+Compound 2a, Compound4+Compound 5+Compound 7, Compound 4+Compound 2a, Compound 4+Compound 2b,Compound 7+Compound 2b, Compound 5+Compound 8, and Compound 5+Compound7.

In still another embodiment, the elevated blood lipid level-relateddisease or disorder is selected from the group consisting of:hyperlipidemia, arteriosclerosis, fatty liver, angina pectoris, stroke,Alzheimer's disease, obesity, diabetes, arthritis, and inflammatorydiseases.

In another aspect, the invention features a method of reducingcholesterol biosynthesis in a subject comprising administering aneffective amount of one or more gallic acid derivatives, therebyreducing cholesterol biosynthesis in a subject.

In another aspect, the invention features a method of increasing thecellular efflux of cholesterol in a subject comprising administering aneffective amount of one or more gallic acid derivatives, therebyincreasing the cellular efflux of cholesterol in a subject.

In another particular aspect, the invention features a method ofinhibiting the cellular uptake of cholesterol in a subject comprisingadministering an effective amount of one or more gallic acidderivatives, thereby inhibiting the cellular uptake of cholesterol in asubject.

In still another aspect, the invention features a method of inhibitingthe oxidation of LDL, comprising administering an effective amount ofone or more gallic acid derivatives, thereby preventing the oxidation ofLDL.

In one embodiment of any one of the above-mentioned aspects, the one ormore gallic acid derivatives is administered as a nutraceutical.

In another embodiment of any one of the above mentioned aspects, the oneor more gallic acid derivatives are selected from the group consistingof: methyl gallate, ethyl gallate, glycerol-1-gallate, glucose-1-gallate(GG1), glucose-6-gallate (GG6), glucose-1,6-digallate (DGG16), mucicacid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone5-gallate, geraniin, corilagin, chebilc acid, and m-digallic acid withminor p-digallic acid.

In a further particular embodiment, the one or more gallic acidderivatives are selected from the group consisting of: Compound4+Compound 5+Compound 2a, Compound 4+Compound 8+Compound 2a, Compound4+Compound 5+Compound 7, Compound 4+Compound 2a, Compound 4+Compound 2b,Compound 7+Compound 2b, Compound 5+Compound 8, and Compound 5+Compound7.

In another embodiment of any of the aspects as set forth above, the atleast one gallic acid derivative is present in an amount from about 10mg-500 mg. In a further related embodiment, the at least one gallic acidderivative is present in an amount from about 40 mg-200 mg.

In another embodiment of any one of the aspects as set forth above, themethod comprises administering at least one or more second agents. In afurther embodiment, the one or more second agents is a therapeuticagent.

In a particular embodiment, the therapeutic agent is selected from thegroup consisting of inhibitors of cholesterol metabolism, inhibitors oftriglyceride synthesis, beta blockers, diuretics, inhibitors of plateletaggregation, angiogenesis inhibitors angiogenesis inhibitors, arthritismedication, toxins, anti-inflammatory agents. In a related embodiment,the therapeutic agent is attached to the gallic acid derivative by acovalent linkage.

In a further embodiment, the therapeutic agent is administered incombination with the one or more gallic acid derivatives.

In another aspect, the invention features a pharmaceutical compositionfor the treatment or prevention of an elevated blood lipid level-relateddisease or disorder comprising one or more gallic acid derivatives and apharmaceutically acceptable excipient.

In one embodiment, the gallic acid derivative is derived from EuMil.

In another embodiment, the one or more gallic acid derivatives isselected from the group comprising: methyl gallate, ethyl gallate,glycerol-1-gallate, glucose-1-gallate (GG1), glucose-6-gallate (GG6),glucose-1,6-digallate (DGG16), mucic acid-2-gallate, 1-methylmucate-2-gallate, mucic acid 1,4-lactone 5-gallate, geraniin, corilagin,chebilc acid, and m-digallic acid with minor p-digallic acid.

In another embodiment, the pharmaceutical composition comprises acombination of gallic acid derivatives selected from the groupconsisting of: Compound 4+Compound 5+Compound 2a, Compound 4+Compound8+Compound 2a, Compound 4+Compound 5+Compound 7, Compound 4+Compound 2a,Compound 4+Compound 2b, Compound 7+Compound 2b, Compound 5+Compound 8,and Compound 5+Compound 7.

In a particular embodiment, the at least one gallic acid derivative ispresent in an amount from about 10 mg-500 mg. In a related embodiment,the at least one gallic acid derivative is present in an amount fromabout 40mg-200 mg.

In another embodiment, the composition further comprises one or moresecond agents. In a related embodiment, the one or more second agents isa therapeutic agent.

In another embodiment, the therapeutic agent is selected from the groupconsisting of inhibitors of cholesterol metabolism, inhibitors oftriglyceride synthesis, beta blockers, diuretics, inhibitors of plateletaggregation, angiogenesis inhibitors, arthritis medication, toxins,anti-inflammatory agents.

In a further embodiment, the therapeutic agent is attached to the gallicacid derivative by a covalent linkage.

In one embodiment of any one of the above-mentioned aspects, thecomposition is administered in a dosage form selected from the groupconsisting of a: food composition, tablet, pill, gel, patch, capsule,suspension tablet, liquid, aqueous emulsion powder, lozenge, sachet,cachet, elixir, suspension, emulsion, syrup, aerosol, ointment, softgelatin capsule, and hard gelatin capsule, suppository, creams, lotions,solutions, gels, pastes powder, and suspension.

In a further embodiment, the food is a health food product, a foodproduct made from cereal flour, gums, a dairy product, a soup, a broth,a paste, a sauce, a beverage, a vitamin complex, a food rich incholesterol, salt, or pepper.

In another embodiment, the dosage form is further selected from thegroup consisting of immediate release, sustained release, and delayedrelease.

In a particular embodiment, the dosage comprises about 0.1% to about 95%gallic acid derivative weight to weight of the composition.

In another embodiment of any one of the above aspects, the subject is amammal. In another related embodiment, the mammal is a human

In another aspect, the invention features a kit for the use inpreventing or treating an elevated blood lipid level-related disease ina mammal comprising one or more gallic acid derivatives.

In one embodiment, the one or more gallic acid derivatives is selectedfrom the group comprising: methyl gallate, ethyl gallate,glycerol-1-gallate, glucose-1-gallate (GG1), glucose-6-gallate (GG6),glucose-1,6-digallate (DGG16), mucic acid-2-gallate, 1-methylmucate-2-gallate, mucic acid 1,4-lactone 5-gallate, geraniin, corilagin,chebilc acid, and m-digallic acid with minor p-digallic acid, andinstructions for use.

In another embodiment, the kit comprises a combination of gallic acidderivatives selected from the group consisting of: Compound 4+Compound5+Compound 2a, Compound 4+Compound 8+Compound 2a, Compound 4+Compound5+Compound 7, Compound 4+Compound 2a, Compound 4+Compound 2b, Compound7+Compound 2b, Compound 5+Compound 8, and Compound 5+Compound 7.

In another aspect, the invention features a kit for the use inpreventing or treating an elevated blood lipid level related disease ordisorder in a subject comprising one or more gallic acid derivatives,and instructions for use.

In another aspect, the invention features a kit for the use inpreventing or treating inflammation in a subject comprising one or moregallic acid derivatives, and instructions for use.

In still another aspect, the invention features a kit for the use inpreventing or treating a stress response in a subject comprising one ormore gallic acid derivatives, and instructions for use.

In another aspect, the invention features a kit for use in reducingcholesterol biosynthesis, increasing the cellular efflux of cholesterol,or inhibiting the cellular uptake of cholesterol comprising one or moregallic acid derivatives, and instructions for use.

In one embodiment of any one of the above-mentioned aspects, the one ormore gallic acid derivatives is administered as a nutraceutical.

In another embodiment of any one of the above-mentioned aspects, the oneor more gallic acid derivatives is selected from the group comprising:methyl gallate, ethyl gallate, glycerol-1-gallate, glucose-1-gallate(GG1), glucose-6-gallate (GG6), glucose-1,6-digallate (DGG16), mucicacid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone5-gallate, geraniin, corilagin, chebilc acid, and m-digallic acid withminor p-digallic acid, and instructions for use.

In another embodiment, the invention features a kit comprising apharmaceutical composition according to any one of the above-mentionedaspects, and instructions for use.

Each of the aspects described herein may be combined with one, more thanone or all of the other aspects and features within each of the aspectsmay be combined with features from the other aspects.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of active compounds isolated from the fruitof Emblica officinalis (EO).

FIG. 2 is a graph that shows the effect of EuMil on the plasma level ofcholesterol. Rabbits were fed a diet including rabbit chow (control),fat fed (14% coconut oil plus 0.2% cholesterol), fat fed plus EuMil (1gm/kg), EuMil alone (1 gm/kg). At 1,2,3 and 4 month intervals blood wasdrawn and plasma levels of lipid/lipoprotein was measured. Values werederived from 3 rabbits in each group.

FIG. 3 is a graph that shows the effect of EuMil on the plasma LDLcholesterol levels in rabbits. Rabbits were fed rabbit chow (control),fat fed (14% coconut oil plus 0.2% cholesterol), fat fed plus EuMil (1gm/kg), EuMil alone (1 gm/kg). Plasma samples were subjected toultracentrifugation to isolate beta-lipoprotein particles(LDL/beta-VLDL). Values were derived from 3 rabbits in each group.

FIG. 4 is a graph that shows the effect of EuMil on the plasmatriglyceride level in rabbits. Rabbits were fed rabbit chow (control),fat fed (14% coconut oil plus 0.2% cholesterol), fat fed plus EuMil (1gm/kg), EuMil alone (1 gm/kg). The plasma samples were subjected totriglyceride assay. Values were derived from 3 rabbits in each group.

FIG. 5 are two graphs that show the effect of EuMil on lipids,lipoproteins and apolipoproteins in human subjects. A 500 mg capsule ofEuMil was swallowed daily for two weeks. Blood was collected byvenipuncture and lipid lipoprotein and apoprotein levels were measuredusing standardized laboratory procedures.

FIG. 6 is a graph that shows the effect of EuMil and its ingredients oncholesterol synthesis in human aortic smooth muscle cells. Cells wereincubated with aqueous extracts of O. sanctum (JH-1), EO (JH-2), W.somnifera (JH-3) and EuMil (JH-4) at concentration 500 μg/ml in mediumsupplemented with 100 μg/ml oxidized LDL. Cells incubated with 100 μg/mlox-LDL served as a control. [14C] acetate (2 μCi/ml) was added andincorporation of radioactivity into cholesterol and cholesteryl esterswas measured after 24 h of incubation in standard cell cultureconditions. Lovastatin (10 μM) plus ox-LDL served as a positive control.

FIG. 7 is a graph that shows typical preparative HPLC chromatogram (UVdetection at 210 nM) of the 1:3 ethanol:water extract of EO fruit powderwith isolated compounds numbered 1 to 9. Also noted are geraniinnumbered 10, corilagin numbered 11, chebulic acid numbered 12 andm-digallic acid numbered 13, mucic acid to gallate derivatives (A),derivatives of the methyl ester of mucic acid gallate (B), derivativesof digalloyl glucose (C) and higher molecular weight tanninsEthylgallate (2b) was not present in the extraction.

FIG. 8 is a graph showing the effect of active fractions from EO oncholesterol synthesis in human aortic smooth muscle cells.

FIG. 9 is a graph showing the effect of active fractions from EO oncholesterol level in human ASMC. Cells were incubated with activefractions (5 μg/ml) and cholesterol mass was determined.

FIG. 10 is a graph showing the effect of active compounds from EO onefflux of cholesterol in human aortic smooth muscle cells. Confluentculture of H-ASMC in 96 well plates was incubated with [3H]cholesterol(5 μCi/ml) for 24 h. Medium was replaced with fresh medium andincubation with herbal fractions was continued for another 24 h. Theincorporation of radioactivity into the cells was measured byscintillation spectrometry (N=6).

FIG. 11 is a graph showing the effect of active compounds from EO onuptake of cholesterol in human aortic smooth muscle cells. Confluentculture of cells grown in 96 well plates was incubated in serum-freemedium with [3H]cholesterol (5 μCi/ml) with and without activefractions. After 24 h the uptake of cholesterol was measured byscintillation spectrometry.

FIG. 12 shows a HPLC chromatogram of the 25% aqueous ethanolic extractof the fruit of E. offcinalis after polyamide treatment (conditions asused for HPLC) C=derivatives of digalloyl glucose, D=derivatives ofgalloyl glucose, E=sugar with some galloyl glucose.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to methods and compositions forreducing hypercholesteremia in a subject. The invention is based on theidentification of novel compounds from crude Embilica officinialis (EO)extracts. The invention relates to the determination of the biologicalactivity and mechanism of action by which the purified compounds lowercholesterol levels and provides nutraceuticals.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. The term “a nucleic acid molecule” includesa plurality of nucleic acid molecules.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements. “Consisting essentially of”, when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

As used herein, the terms “therapeutically effective” or “amountsufficient” refers to when a composition or method of the invention isproperly administered in vivo to a mammal, including humans, ameasurable beneficial effect occurs. Exemplary beneficial effectsinclude measurable reduction in the level of cholesterol and/or LDLand/or triglycerides in the blood of the mammal; reduction of clinicallyverifiable and/or patient-reported level of high cholesterol and/or LDLand/or triglycerides or complete resolution or curing of the elevatedLDL and/or cholesterol and/or triglyceride condition or other diseases.

As used herein, the phrase “elevated lipid level-related disease ordisorder” is meant to refer to any disease or disorder that ischaracterized by or is related to an elevated level of LDL or withelevated levels of total cholesterol. In certain preferred embodiments,an elevated blood lipid level related disease or disorder can be, but isnot limited to, hyperlipidemia, arteriosclerosis, fatty liver, anginapectoris, stroke, alzheimer's disease, obesity, diabetes, arthritis, andinflammatory diseases.

The terms “hypercholesteremia” or “hypercholesterolemia” as used hereinmean the presence of high levels of cholesterol in the blood of amammal.

As used herein, the terms “concurrent” or “concurrent administration”mean that the EO composition and an agent, in certain embodiments atherapeutic agent, are administered to the subject either (a)simultaneously in time (optionally by formulating the two together in acommon carrier), or (b) at different times during the course of a commontreatment schedule. In the latter case, the two compounds areadministered sufficiently close in time to achieve the intended effect.

As used herein, the term “gallic acid derivative” is meant to refer to aderivative of 3,4,5-Trihydroxybenzoic acid. In certain preferredembodiments, the gallic acid derivative is isolated from the fruit fromEmbilica officinalis (EO). In other preferred embodiments, the gallicacid derivatives are selected from compounds 2a-13.

As used herein, the term “stress response” is meant to refer to theability of one or more gallic acid derivatives to lower the expressionof a protein that is a marker of stress, for example, but not limitedto, a heat shock protein. In certain embodiments, a stress response ismeasured by the ability of one or more gallic acid derivatives to lowerthe expression of heat shock protein 70 (Hsp70).

As used herein, the term “cholesterol efflux” is meant to refer to theability of a compound to promote the release of cholesterol from cells.

As used herein, the term “EuMil” is meant to refer to a polyherbalformulation consisting of extracts of Emblica officinalis Gaertn (syn.Phyllanthus emblica Linn.) (EO), Withania somnifera (L) Dunal and Ocimumsanctum L with Asparagus racemosus Willd used as filler. In preferredembodiments, the extracts of EO, Withania somnifera (L) Dunal and Ocimumare used in equal portions.

As used herein, the phrase “in combination with” is intended to refer toall forms of administration that provide an inhibitory nucleic acidmolecule together with a second agent, such as a second inhibitorynucleic acid molecule or a chemotherapeutic agent, where the two areadministered concurrently or sequentially in any order.

As used herein, a “subject” is a vertebrate, preferably a mammal, morepreferably a human. Mammals include, but are not limited to, murines,simians, humans, farm animals, sport animals, zoo animals and pets, forexample cats, dogs, cattle, fish, birds. A subject can include agenetically compromised or genetically manipulated vertebrate,preferably a genetically compromised or genetically manipulated mammal,more preferably genetically compromised or genetically manipulated farmanimals, sport animals, zoo animals or pets, for example cats, dogs,cattle, fish, birds.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid filler, diluents or encapsulatingsubstances that are suitable for administration into a human

The term “nutraceutical” is meant to refer to any substance that is afood or a part of a food or something that is added to a food, andprovides medical or health benefits, including the prevention and/ orthe treatment of a disease or disorder. Nutraceuticals are often naturalproducts that are identified from botanicals, and purified or partiallypurified and then included in foods.

I. Cholesterol

Cholesterol is the principal sterol of humans and higher animals.Cholesterol is an important component of cell membranes andlipoproteins, and is a key biosynthetic precursor of bile acids andsteroidal hormones. Cholesterol is found in all body tissues and alsoamong the lipids in the bloodstream, but with especially high content inthe brain, spinal cord and in animal fats. Nevertheless, an increasedlevel of cholesterol in the blood, especially in its low-densitylipoprotein-bound form, is the critical factor in the development ofcoronary heart disease, which creates a danger of heart attack.

Because of insolubility of cholesterol, it is transported in the bloodin a modified form of lipoproteins. There are a number of lipoproteinsdiffering in their properties and physiological role, but the mostimportant are low-density lipoprotein (LDL) and high-density lipoprotein(HDL).

LDL is a major carrier of cholesterol in the blood and, as mentionedabove, elevated LDL levels are a major risk factor in the development ofcoronary heart disease. Its excess in the blood leads to a situationwhen arteries are blocked to a greater or lesser extent by thedeposition of cholesterol plaques that leads to a condition of stenosisor atherosclerosis in particular. Inflammation, hemodynamic factors andthe like contribute to plaque rupture that, in turn, further contributesto thrombus formation, blocking blood flow in coronary or cerebralarterial vessels.

The following approximate values are generally considered optimal, nearoptimal, borderline, high and very high:

LDL Cholesterol (mg/dL) <100 Optimal 100-129 Near optimal/above optimal130-159 Borderline high 160-189 High >=190 Very high Total Cholesterol(mg/dL) <200 Desirable 200-239 Borderline high >=240 High HDLCholesterol (mg/dL) <40 Low >=60 High

The above recommendations are for individuals with moderate to highrisk. Persons with very high risk such as established CVD plus multiplemajor risk factors, severe and poorly controlled risk factors, multiplerisk factors of metabolic syndrome, acute coronary syndromes the goal isto have LDL-cholesterol below 70 mg/dL.

Typically, the average person consumes between 350-400 mg of cholesteroldaily, while the recommended intake is around 300 milligrams. Increaseddietary cholesterol consumption, especially in conjunction with a diethigh in saturated fat intake, can result in elevated serum cholesterol.Having an elevated serum cholesterol level is a well-established riskfactor for heart disease, and therefore there is a need to mitigate theundesired effects of cholesterol accumulation. High cholesterol levelsare generally considered to be those total cholesterol levels at 200milligrams and above, or LDL cholesterol levels at 130 milligrams andabove. By lowering the total system LDL cholesterol level, it isbelieved that certain health risks, such as coronary disease andpossibly some cancers that are typically associated with highcholesterol levels, can be reduced.

Numerous studies relating to modifying the intestinal metabolism oflipids illustrate that such effects can reduce a high cholesterol level(Burnett et al., Expert Opin Investig Drugs., 5(11):1337-51, 2006; Wanget al., Am J Physiol Gastrointest Liver Physiol., 287(3):G547-54, 2004;Heidrich et al., BMC Pharmacology, 4:5, 2004; Borel et al., Am. J. Clin.Nutr., 49:1192-1202, 1989; Malinow et al., Am. J. Clin,. Nutr.,30:2061-2067, 1977). Hampering the absorption of triglycerides,cholesterol or bile acids, or a combination of these mechanisms, resultsin a lowering of cholesterol levels in the serum (Lewis et al., Journalof Lipid Research, 36:1098-1105, 1995). Also by interfering with thedigestion of dietary phospholipids these compounds may affect a criticalmicelle formation and consequently the absorption of cholesterol.

Seminal studies by Steinberg et al. have demonstrated that LDL andox-LDL are cytotoxic to cells of the vascular wall and may contribute toendothelial injury (23). The entry of ox-LDL into the sub-endothelialspace and uptake by monocytes and aortic smooth muscle cells contributeto fatty streak formation. Alternatively, LDL taken up via pinocyticmechanism by the vascular endothelial cells allows LDL to enter thesub-endothelial space. Here depending upon the anti-oxidant status/levelof the vascular wall the LDL may undergo oxidation. Such minimallyoxidized LDL or fully ox-LDL is taken up by monocytes/smooth musclecells leading to foam cell formation a “hallmark in the pathogenesis inatherosclerosis” (24). In addition, oxidized phospholipids componentspresent in minimally oxidized lipoproteins such as 1-palmitoyl2-(5-oxovaleroyl) phosphatidylcholine (POVPC) can stimulate theproliferation of aortic smooth muscle cells, contributing tointima/media thickening (25).

Human plasma LDL plays a major role in the delivery of cholesterol fromhepatic tissue to extra-hepatic tissue through the LDL (Beta) receptor[3]. Nonetheless, elevated levels of plasma and LDL cholesterol promoteatherosclerosis via an increased uptake through a LDLreceptor-independent pathway. For example, previous studies have shownthat modified LDL, upon oxidation or glycosylation, has a predilectionfor uptake through the LDL receptor-independent pathway. Thus ox-LDLtaken by aortic smooth muscle cells and macrophages through the LDLreceptor independent/scavenger pathway contributes to foam cell/fattystreak formation by stimulating cholesteryl ester accumulation and byinhibiting its degradation [4].

When the flow is directed to a part of the heart muscle, this can causea heart attack. If a thrombus blocks the blood flow to a certain regionof the brain, the consequence is a stroke.

In contrast to LDL, HDL carries the excess of cholesterol from tissuesaway to the liver. It is considered to be able to remove somecholesterol from atherosclerotic plaques thus making their growthslower. Angiographical studies showed a correlation between elevatedlevels of HDL and a decreased number of sites of stenosis in thecoronary arteries of humans. This indicates a protective action of HDLagainst heart attack and indicates a possibility to use measuring HDLlevel as a prognostic indicator of higher or lower risk.

Demand of the body in cholesterol is covered by two sources: by thebiosynthesis that mainly proceeds in the liver, intestine and skin, andvia uptake from food, mainly from animal and dairy products. Under mixeddiet, ratio of cholesterol amounts supplied by the sources is about 1:1.

Cellular cholesterol homeostasis is very important for the prevention ofcoronary heart disease. In general, the plasma concentration ofcholesterol in the body is regulated by the dietary cholesterolabsorption, by the biosynthesis of cholesterol itself and its esterifiedforms, by the metabolic removal of circulating cholesterol, and by theexcretion of cholesterol via bile and feces, by cholesterol efflux fromperipheral tissues via HDL and back to the liver for further metabolism.

Both diet and genetically determined biosynthetic-metabolic specificityof the body are instrumental in the development of atherosclerosis. Adiet high in cholesterol will lead to a high level of cholesterol in thebloodstream, which has important consequences. Dietary cholesterolsuppresses the biosynthesis of cholesterol in the body, especially intissues other than the liver. A parallel effect is inhibition ofsynthesis of LDL receptors. As a result of reduction in the number ofreceptors, the level of LDL in blood increases, leading to thedeposition of atherosclerotic plaques. Damaged biosynthesis of LDLreceptors could be also a result of a genetic deviation. Thus, a goodregulation of cholesterol biosynthesis is very important. Oxygenatedderivatives of cholesterol seem to control the biosynthesis of theresponsible enzymes in a receptor-mediated process, providing a feedbackregulation for the biosynthesis of cholesterol. Thus, modern approachesto prevention of atherosclerosis are based on the correction of bothexternal and internal factors ruling the cholesterol level in blood:dietary supply and absorption of exogenic cholesterol, on the one hand,and the biosynthesis of endogenic cholesterol and related structures, onthe other hand.

Statins

In correcting internal factors, inhibitors of cholesterol (and itsderivatives) biosynthesis play an important role and the search for newagents with this activity now constitutes a major research effort. For anumber of years, significant research went into the development ofcompetitive inhibitors for 3-hydroxy-3-methylglutaryl coenzyme Areductase, a major regulatory enzyme of cholesterol biosynthesis. Manyattempts to use oxygenated sterols for this purpose, which via bindingoxysterol receptors were expected to decrease activity of HMG-CoAreductase, did not bring practical results. A breakthrough came with thediscovery of a series of fungal metabolites with very high affinitiesfor HMG-CoA reductase which were found to be highly efficient inhibitorsof cholesterol biosynthesis. Nowadays these compounds and some syntheticanalogs, commonly known as statins, are available commercially andwidely used.

Although being relatively safe and efficient in treatment and preventionof coronary heart disease, statins have certain limitations in their useand they need care in application because of possible side effects suchas those provided below.

In mammalian cells, the rate-limiting step in cholesterol biosynthesisinvolves the conversion of hydroxymethylglutaryl co-enzyme A (HMG-CoA)to mevalonic acid through the enzyme HMG-CoA reductase. The widelyprescribed statin, simvastatin (ZOCOR), inhibits HMG-CoA reductaseactivity and markedly reduces the blood levels of cholesterol as well asepisodes of stroke and heart attacks [5, 6]. In as much as thecollective group of statins can lower blood level of cholesterol, theymay impart several adverse effects such as muscle weakness and renalfailure, and are contra-indicated in pregnant women and patients withliver disorders. Thus, patients taking them respond very often to thelowering of cholesterol biosynthesis by a compensatory enhancement ofcholesterol absorption from food and, especially for the cases whenstatins are used as a monotherapy, some patients fail to reach treatmentgoals. The risk of liver complications dictates the use of statins undermedical control. Taking additionally into account the relatively highcosts of statin therapy, which varies from $20,000 to $40,000 perquality-adjusted life-year saved [John A. Farmer, Economic Implicationsof Lipid-Lowering Trials: Current Considerations in Selecting a Statin,Am J Cardiol 1998; 82:26M31M] and the desirability of long-termpermanent treatment [Terry A. Jacobson, Clinical Context: CurrentConcepts of Coronary Heart Disease Management, Am J Med. 2001; 110(6A):3S-11S], it is evident that the new agents are needed with similartargeting as the statins but with higher potency, safety, availabilityand significant cost savings for patients, third party insurers, stateand federal government payors as well.

There also exists a significant patient population in whom statins arenot effective, so the need for an agent that will lower cholesterolextends beyond the number of patients currently taking statins to lowertheir cholesterol levels.

Other Cholesterol Modulating Agents

The uptake of dietary cholesterol by intestine and other tissues as wellthe cellular efflux of cholesterol also are key determinants ofcholesterol homeostasis [7,8]. Accordingly, drugs that can collectivelymodulate multiple cholesterol homeostatic mechanisms have becomeavailable, such as VYTORIN, which is a mixture of ZETIA andZOCOR/SIMVASTATIN. ZETIA inhibits the uptake of dietary cholesterol inintestinal epithelial cells while Simvastatin inhibits the activity ofHMG-CoA reductase, that in turn further inhibits endogenous cholesterolbiosynthesis. However, these drugs also have numerous side effects.

Torcetrapib an inhibitor of cholesterol ester transfer protein (CETP) isanother class of compounds that lower blood levels of cholesterol byraising HDL cholesterol levels. CETP plays a role in the reversecholesterol transport pathway as well as in the intravascular remodelingand recycling of HDL particles. CETP promotes the transfer ofcholesteryl esters from beneficial antiatherogenic HDLs toproatherogenic apolipoprotein B-containing lipoproteins (e.g., LDL andvery low-density lipoprotein cholesterol). When there is an excess ofcirculating concentrations of atherogenic lipoproteins relative to HDL,arterial cholesterol deposition and atherogenesis are greatly enhanced.Thus, high plasma levels of CETP are correlated with low HDL cholesterollevels. However, Torcetrapib has been withdrawn from the market becauseof deleterious effects in man. In general, the concept of CETPinhibitors as useful candidates is still viable and may be of use as atarget to lower cholesterol in the invention as described herein.

Elevated Blood Lipid Level Related Diseases

The invention features, in certain embodiments, methods for preventingor treating an elevated blood lipid level-related disease or disorder.In certain embodiments, an elevated lipid level-related disease ordisorder can refer to, but is not limited to, any disease or disorderthat is characterized by or is related to an elevated level of LDL orwith elevated levels of total cholesterol.

In certain preferred embodiments, an elevated blood lipid level relateddisease or disorder can be, but is not limited to, hyperlipidemia,arteriosclerosis, fatty liver, angina pectoris, stroke, Alzheimer'sdisease, obesity, diabetes, arthritis, and inflammatory diseases.

Although not limited to the following, preferred elevated lipidlevel-related diseases or disorders that can be treated by the methodsof the invention include hypercholesterolemia, mixed hyperlipidemias,metabolic syndrome, hypo-alphaprotienemia, myocardial infarction,stroke, Alzheimer's disease, diabetes, and obesity, and post menopausalwomen.

Hypercholesteremia

Hypercholesterolemia is an important risk factor definitively connectedwith cardiovascular disease and, particularly, with atherosclerosis andcoronary heart disease. Millions of people around the world suffer fromcoronary heart disease, which is the leading cause of death andmorbidity in a productive age, especially in Western Europe and in theUnited States. For this reason it is also a significant drain onhealthcare resources in the Western world. For example, in the USA totalcosts (direct and indirect) connected with the disease were estimated tobe about $118 billion in 2000. For 1.1 million citizens that experiencedmyocardial infarction, more than 40% of those died [Terry A. Jacobson,Clinical Context: Current Concepts of Coronary Heart Disease Management,Am J Med. 2001; 110 (6A):3S11S]. In addition, this disease is growing atan alarming rate in Asian countries particularly among Asian Indianswhere CVD has reached epidemic proportions [1]. Several established riskfactors that contribute to CVD include elevated levels of bloodcholesterol, homocysteine, high sensitive C-reactive proteins (hs-CRP),diabetes, stroke and stress [2].

Atherosclerosis

Atherosclerosis is the most common cause of death and serious morbidityin the Western world. Atherosclerosis is one of three morphologicallydistinct forms of arteriosclerosis. Arteriosclerosis is the hardening ofthe arteries due to their thickening and loss of elasticity.Atherosclerosis occurs when irregularly distributed lipid deposits inthe inner coating of the vessels of the elastic arteries, such as theaorta, carotid and iliac, or the large and medium-sized musculararteries, such as the coronary and popliteal. These lipid deposits,called atheromatous plaques, cause fibrosis and calcification, whichleads to coronary heart disease and myocardial infarction. The plaquesare comprised of cells, macrophages and other leukocytes, a connectivetissue extra-cellular matrix and intracellular and extracellular lipiddeposits. The progression of atherosclerosis can be slowed by reducingthe plasma cholesterol and cholesterol LDL levels.

Cholesterol and its oxidized derivatives are thought to accumulate inatherosclerotic lesions when cholesterol influx exceeds efflux. This mayprovide an explanation for atherosclerosis in patients with lipiddisorders.

Correcting external factors is realized via diet modification, such asreduction of dietary supply of cholesterol, for example by partialsubstitution of food animal fats by plant fats that do not containcholesterol. The reduction of dietary cholesterol absorption can bereached via application of special food additives or foods enriched bythe abundant phytosterols, such as beta-sitosterol or campesterol, ortheir saturated derivatives (stanols). Plant sterols produceanticholesterolemic effect which is considered to be connected with theinhibition of cholesterol absorption in the intestine because ofcompetition with cholesterol for incorporation into micelles, althoughother absorption steps may also be involved. When the plant sterolsreplace cholesterol of the micelles, free cholesterol is excreted withfeces. A limitation of the approach is that relatively large doses ofsterols are required for modest reduction in plasma cholesterol. Thesame is true in respect to other agents blocking cholesterol absorption,such as stanols, aminoglycoside, the antibiotic neomycin, which appearsto inhibit cholesterol absorption by forming complexes with cholesterolthat are excreted, and the bile salt binder cholestyramine, an anionexchanger that indirectly alters cholesterol levels by limiting theresorption of cholesterol-derived bile salts.

Hyperlipidemia

Hyperlipidemia is an elevation of lipids (fats) in the bloodstream.These lipids include cholesterol, cholesterol esters (compounds),phospholipids and triglycerides. When hyperlipidemia is defined in termsof a class or classes of elevated lipoproteins in the blood, the termhyperlipoproteinemia is used. Hypercholesterolemia is the term for highcholesterol levels in the blood. Hypertriglyceridemia refers to hightriglyceride levels in the blood. The American Heart Associationprovides information to the public on hyperlipidemia on the world wideweb at americanheart.org/presenter.jhtml?identifier=4600.

Included are hyperlipidemias that are induced by other conditions oragents, for example HIV drug inducedhypercholesterolemia/hyperlipidemia.

Fatty Liver

Fatty liver refers to the build-up of excess fat in the liver cells. Itis normal for the liver to contain some fat; however, if fat accountsfor more than 10% of your liver's weight, a subject is considered tohave fatty liver and is at a risk for developing more seriouscomplications.

Fatty liver may cause no damage, but sometimes the excess fat leads toinflammation of the liver, which results in liver damage. Thiscondition, called steatohepatitis, does cause liver damage. Sometimes,inflammation from a fatty liver is linked to alcohol abuse; this isknown as alcoholic steatohepatitis. Otherwise the condition is callednonalcoholic steatohepatitis, or NASH. A fatty liver produces nosymptoms on its own, and people often learn about their fatty liver whenthey have medical tests for other reasons. NASH can damage the liver foryears or even decades without causing any symptoms.

Eating excess fat and/or calories causes fat to build up in the liver.When the liver does not process and break down fat as it normallyshould, too much fat will accumulate. People tend to develop fatty liverif they have certain other conditions, such as obesity, diabetes, orhigh triglycerides. Alcohol abuse, rapid weight loss and malnutritionmay also lead to fatty liver. However, some people develop fatty livereven if they have none of these conditions. More information on fattyliver disease is available publicly on the world wide web atliverfoundation.org/education/info/fattyliver/.

Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia among olderpeople. AD begins slowly. It first involves the parts of the brain thatcontrol thought, memory and language. AD usually begins after age 60.The risk goes up as you get older. Your risk is also higher if a familymember has had the disease. Currently, no treatment can stop thedisease.

A hallmark of all forms of Alzheimer's disease (AD) is an abnormalaccumulation of the -amyloid protein (A) in specific brain regions. Boththe generation and clearance of A are regulated by cholesterol. Elevatedcholesterol levels increase A in cellular and most animal models of AD,and drugs that inhibit cholesterol synthesis lower A in these models.The identification of a variant of the apolipoprotein E (APOE) gene as amajor genetic risk factor for AD is also consistent with a role forcholesterol in the pathogenesis of AD. Thus, lowering neuronalcholesterol levels is a strategy for treating and preventing AD. Areview by Simons et al. (Neurology 2001. 2001 Sep. 25; 57(6):1089-93.)discusses a link between cholesterol and AD, and discusses howcholesterol might modulate Abeta deposit formation.

Diabetes

People with diabetes are more likely to suffer from, and die from,cardiovascular problems (like a heart attack or stroke) than thosewithout diabetes. Diabetes can upset the balance between HDL and LDLlevels. People with diabetes tend to have LDL particles that stick toarteries and damage their walls more easily. Glucose latches ontolipoproteins, and these glucose coated LDL particles remain in theblood-stream longer and may lead to plaques. People with diabetes tendto have low HDL and high triglyceride levels, both of which boost therisk of heart and artery disease. As a result, in people with diabetes:heart disease occurs earlier, heart disease is two to four times ascommon, and heart disease is more often fatal. Accordingly, heartdisease in people with diabetes is a major public health problem, onethat is expected to get worse. A Centers for Disease Control andPrevention study estimated that one-third of babies born in 2000 willsomeday develop diabetes. People can reduce their risk of heart andblood vessel disease by lowering their cholesterol levels.

Metabolic Syndrome

Metabolic syndrome is a disease or disorder wherein the patient has highLDL cholesterol, low HDL cholesterol and high triglyceride levels inblood. People with metabolic syndrome are at increased risk of coronaryheart disease and other diseases related to plaque buildups in arterywalls (e.g., stroke and peripheral vascular disease) and type 2diabetes. The metabolic syndrome has become increasingly common in theUnited States, and it is estimated that over 50 million Americans haveit.

Metabolic syndrome can be characterized by a group of metabolic riskfactors that include: abdominal obesity (excessive fat tissue in andaround the abdomen); atherogenic dyslipidemia (blood fat disorders—hightriglycerides, low HDL cholesterol and high LDL cholesterol—that fosterplaque buildups in artery walls); Elevated blood pressure; Insulinresistance or glucose intolerance (the body can't properly use insulinor blood sugar); Prothrombotic state (e.g., high fibrinogen orplasminogen activator inhibitor-1 in the blood); Proinflammatory state(e.g., elevated C-reactive protein in the blood).

Lowering LDL cholesterol to recommended levels is among the first linesof therapeutic aims in treating the metabolic syndrome.

Hypo-Alphaproteinemia

Hypo-alphaproteinemia is a disease where the patient has the inabilityto synthesize sufficient amount of HDL and or catabolizes HDL rapidly.

II. Eu Mil

EuMil is a polyherbal formulation consisting of equal portions ofstandardized extracts of Emblica officinalis Gaertn (syn. Phyllanthusemblica Linn.) (EO), Withania somnifera (L) Dunal and Ocimum sanctum Lwith Asparagus racemosus Willd used as filler [9-11]. Although EuMil isusually prescribed as an anti-stress and performance enhancing herbalmedicine, little is known about its hypocholesterolemic properties. Forinstance, in a study by Bhattacharya et al. (Indian J Exp Biol. 2002October; 40(10):1161-3) examined anti-stress activity of EuMil, butprovided no teaching or suggestion of any anti-cholesteremic activity.Here, applicants have shown that EuMil has hypocholesterolemicproperties in a formulation fed to rabbits with experimentalhyperlipidemia, (U.S. Provisional Application 60/876,761 filed Dec. 22,2006, U.S. Provisional Application 60/877,753 filed Dec. 29, 2006, U.S.Provisional Application 60/876,599 filed Dec. 22, 2006 and U.S.Provisional Application 60/877,740 filed Dec. 29, 2006, all incorporatedby reference herein). Further research has shown that the only componentof EuMil that had significant hypocholesterolemic activity was thepowdered fruit of EO.

The fruit of EO (Amla in Hindi) is widely used in India, Sri Lanka,Pakistan, Uzbekistan, South East Asia and China as a traditionalmedicine to treat many diseases such as diarrhea, jaundice, dyspepsiaand hemorrhage amongst others [12-14]. The activity of Amla usually hasbeen ascribed to its high content of vitamin C, which is stabilized inthe fruit due to presence of tannins [15,16].

Previous Ayurvedic studies have suggested that several herbal mixturesexert their effect in a synergistic manner Moreover, since EuMil is madeup of a admixture of EO, W. somnifera and O. sanctum (with Asparagusracemosus used as a filler for pill preparation), the instant inventionis focused on whether this herbal preparation exerted itshypocholesterolemic effect in a synergistic or additive manner (e.g. asa mixture) or whether the activity was confined to only one of the aboveherbal ingredients.

III. Compounds

As described herein, EuMil is made up of a mixture of EO, W. somniferaand O. sanctum (with Asparagus racemosus used as a filler for pillpreparation).

The invention features in certain embodiments isolated fractions ofEuMil as set forth below, and numbered as compounds (1), (2a), (2b),(3,) (4), (5), (6), (7), (8), (9), (10), (11), (12) and (13).

In certain embodiments, the invention features administering to asubject an effective amount of one or more gallic acid derivatives. Inmore specific examples, the gallic acid derivatives are selected fromthe compounds as described above, compounds 2 (a) and (b)-13.

In other certain embodiments, the compound used is compound 4.

In other certain embodiments, the compound used is compound 5.

In other certain embodiments, the compound used is compound 7.

In other certain embodiments, the compound used is compound 8.

In other certain embodiments, the compound used is compound 9.

In certain preferred embodiments, the first compound of choice will becompound 4, the second most preferred will be compound 5, the third mostpreferred will be compound 7, the third most preferred will be compound8, the fourth most preferred will be compound 9.

In certain embodiments, the one or more gallic acid derivatives arecombined, for example combinations of two, three or more gallic acidderivatives. The gallic acid derivatives may be combined as one or moreof a cholesterol lowering gallate derivative with one or more of acholesterol uptake/absorption inhibitor.

In certain preferred embodiments, the gallic acid derivatives arecombined as, but not limited to the following combinations:

Compound 4+Compound 5+Compound 2a

Compound 4+Compound 8+Compound 2a

Compound 4+Compound 5+Compound 7

Compound 4+Compound 2a

Compound 4+Compound 2b

Compound 7+Compound 2b

Compound 5+Compound 8

Compound 5+Compound 7

Gallic acid (Compound 1) has been shown to have to toxic effects. Forexample, in a 2001 report (Food and Chemical Toxicology Volume 39, Issue11, November 2001, Pages 1063-1070), Shibutani et al. reported thetoxicity of gallic acid (GA) was investigated in F344 rats by feeding adiet containing 0, 0.2, 0.6, 1.7 and 5% GA for 13 weeks. Toxicologicalparameters included clinical signs, body weight, food consumption,hematology, blood biochemistry, organ weights and histopathologicalassessment. Body weight gain in the 5% GA-treated animals of both sexesfrom week 1 to the end of the experiment was significantly lower thanthat of the untreated controls. Toxic effects following administrationof 0.6% or more in males and 5% in females included reduction ofhemoglobin concentration, hematocrit and red blood cell counts andincrease in reticulocytes. Histopathologically, extramedullaryhematopoiesis, hemosiderin deposition and congestion appeared in thespleens of 5% GA-treated animals, suggesting development of hemolyticanemia. In addition, centrilobular liver cell hypertrophy, reflected inincrease in liver weight, was observed in animals of both sexes.

Gallic acid has been shown to have apoptotic effects. For example,reports by Qui X. et al (Heart and Vessels 15: 90-99(2000)), Inoue, M(BBRC 204: 898-904,(1994)), Serrano A et al. (Archiv. Biochem. Biophys.350: 49-54 (1995)), Hsu, C L et al. (J. Agric. Food. Chem 55: 354-7356(2007)) show that apoptosis is a mechanism of action of gallic acid.

IV. Methods of Treatment

The invention provides various methods of using the compounds of theinvention for disease therapy. Generally, the compounds of the inventionas described herein are useful to treat an elevated blood lipidlevel-related disease or disorder (e.g. hypercholesteremia) as well asfor providing a delivery vehicle for therapeutic agents (including, butnot limited to, cholesterol-lowering agents, triglyceride-loweringagents and other lipid-lowering agents).

In one aspect, the invention features methods for preventing or treatingan elevated blood lipid level-related disease or disorder in a subjectcomprising administering to the subject an effective amount of one ormore gallic acid derivatives, and thereby preventing or treating anelevated blood lipid level-related disease or disorder in the subject.Blood lipid level related diseases and disorders, as discussed in moredetail herein, can be selected from, but not limited to hyperlipidemia,arteriosclerosis, fatty liver, angina pectoris, stroke, alzheimer'sdisease, obesity, diabetes, arthritis, and inflammatory diseases.

The invention also features methods for preventing or treatinginflammation or a stress response in a subject comprising administeringto the subject an effective amount of one or more gallic acidderivatives, thereby preventing or treating inflammation in the subject.

Inflammation is associated with a large collection of mediators thatinitiate the inflammatory response, recruit and activate other cells tothe site of inflammation, and subsequently resolve the inflammation(Gallin and Snyderman, 1999, Overview in INFLAMMATION: BASIC PRINCIPLESAND CLINICAL CORRELATES, 3 d ed., Lippincott Williams & Wilkins,Philadelphia, pp. 1 3). Hypercholesterolemia has been associated withhigh plasma levels of inflammation-sensitive plasma proteins (ISP). In astudy done by Engstrom et al. (Circulation. 2002; 105:2632-2637), Plasmacholesterol and 5 inflammation-sensitive plasma proteins (ISP)(fibrinogen, alpha 1-antitrypsin, haptoglobin, ceruloplasmin, andorosomucoid) were determined in 6063 healthy men, 28 to 61 years of age.The incidence of stroke, cardiac events (fatal and nonfatal), andcardiovascular deaths was compared between groups defined by levels ofcholesterol and ISP. High cholesterol was associated with higher levelsof ISP. Hypercholesterolemia (6.5 mmol/L, 251 mg/dL) was associated withan increased incidence of ischemic stroke and cardiac events and with areduced incidence of intracerebral hemorrhage. The ISP levels modifiedthese associations. After risk factor adjustment, men withhypercholesterolemia and high ISP levels had a significantly higher riskof cardiovascular death, cardiac events, and ischemic stroke than menwith normal cholesterol and low ISP levels. In the absence of high ISPlevels, hypercholesterolemia was associated with a moderately higherrisk of cardiovascular death and cardiac events, but not significantlywith ischemic stroke.

Elevated stress has been associated with lipid-level related disorders.Macrophages are the most prominent cell type in atherosclerotic lesions.They are present throughout the process of lesion development andcharacteristically accumulate ‘free’ cholesterol—an unesterified form ofcholesterol. This accumulates in endoplasmic reticular (ER) membranes,which are normally devoid of cholesterol. Such abnormal accumulation of‘free’ cholesterol has several adverse effects on ER function, andultimately results in a stress response.

Additionally, work by Werstuck et al. (J Clin Invest, May 2001, Volume107, Number 10, 1263-1273) has shown that homocysteine-inducedendoplasmic reticulum (ER) stress activates both the unfolded proteinresponse and the sterol regulatory element-binding proteins (SREBPs) incultured human hepatocytes as well as vascular endothelial and aorticsmooth muscle cells, and that activation of the SREBPs is associatedwith increased expression of genes responsible forcholesterol/triglyceride biosynthesis and uptake and with intracellularaccumulation of cholesterol.

As discussed in more detail herein the one or more gallic acidderivatives is, in certain preferred embodiments, administered as anutraceutical.

As discussed in more detail herein the one or more gallic acidderivatives are, in other certain preferred embodiments, selected fromthe group consisting of: methyl gallate, ethyl gallate,glycerol-1-gallate, glucose-1-gallate (GG1), glucose-6-gallate (GG6),glucose-1,6-digallate (DGG16), mucic acid-2-gallate, 1-methylmucate-2-gallate, mucic acid 1,4-lactone 5-gallate, geraniin, corilagin,chebilc acid, and m-digallic acid with minor p-digallic acid.

As described herein, the one or more gallic acid derivatives can be acombination of compounds. In certain embodiments, the gallic acidderivatives may be combined as one or more of a cholesterol loweringgallate derivative combined with one or more of a cholesteroluptake/absorption inhibitor. Accordingly, in certain examples, the oneor more gallic acid derivatives are selected from, but not limited to,combinations of: Compound 4+Compound 5+Compound 2a, Compound 4+Compound8+Compound 2a, Compound 4+Compound 5+Compound 7, Compound 4+Compound 2a,Compound 4+Compound 2b, Compound 7+Compound 2b, Compound 5+Compound 8,and Compound 5+Compound 7.

The invention also features methods of affecting (reducing orincreasing) cholesterol metabolism. By cholesterol metabolism is meantcholesterol biosynthesis, cholesterol cellular efflux and/or cholesterolcellular uptake. In particular, the invention features methods ofreducing cholesterol biosynthesis in a subject comprising administeringan effective amount of one or more gallic acid derivatives, therebyreducing cholesterol biosynthesis in a subject.

The invention features methods of increasing the cellular efflux ofcholesterol in a subject comprising administering an effective amount ofone or more gallic acid derivatives, thereby increasing the cellularefflux of cholesterol in a subject.

The invention features methods of inhibiting the cellular uptake ofcholesterol in a subject comprising administering an effective amount ofone or more gallic acid derivatives, thereby inhibiting the cellularuptake of cholesterol in a subject.

The invention also features methods of inhibiting the oxidation of LDLcomprising administering an effective amount of one or more gallic acidderivatives, thereby preventing the oxidation of LDL.

As discussed in more detail herein the one or more gallic acidderivatives is, in certain preferred embodiments, administered as anutraceutical.

As discussed in more detail herein the one or more gallic acidderivatives are, in other certain preferred embodiments, selected fromthe group consisting of: methyl gallate, ethyl gallate,glycerol-1-gallate, glucose-1-gallate (GG1), glucose-6-gallate (GG6),glucose-1,6-digallate (DGG16), mucic acid-2-gallate, 1-methylmucate-2-gallate, mucic acid 1,4-lactone 5-gallate, geraniin, corilagin,chebilc acid, and m-digallic acid with minor p-digallic acid.

As described herein the one or more gallic acid derivatives can be acombination of compounds. In certain embodiments, the gallic acidderivatives may be combined as one or more of a cholesterol loweringgallate derivatives combined with one or more of a cholesteroluptake/absorption inhibitor. Accordingly, in certain examples, the oneor more gallic acid derivatives are selected from, but not limited to,combinations of: Compound 4+Compound 5+Compound 2a, Compound 4+Compound8+Compound 2a, Compound 4+Compound 5+Compound 7, Compound 4+Compound 2a,Compound 4+Compound 2b, Compound 7+Compound 2b, Compound 5+Compound 8,and Compound 5+Compound 7. In any of the methods as described, one ormore second agents can be administered along with the one or more gallicacid derivatives.

V. compositions

As reported herein, one or more gallic acid derivatives has effects oncholesterol synthesis, cholesterol level and/or cholesterol metabolism(e.g. efflux, uptake). Accordingly, in certain examples, the inventionfeatures a pharmaceutical composition for the treatment or prevention ofan elevated blood lipid level-related disease or disorder comprising oneor more gallic acid derivatives and a pharmaceutically acceptableexcipient.

The compositions should be sterile and contain a therapeuticallyeffective amount of the polypeptides or nucleic acid molecules in a unitof weight or volume suitable for administration to a subject. A gallicacid derivative may be administered within pharmaceutically-acceptablediluents, carriers, or excipients, in unit dosage form. Conventionalpharmaceutical practice may be employed to provide suitable formulationsor compositions to administer the compounds to patients suffering fromthe disease or disorder to be treated, for example a disease or disorderof elevated lipid level. Administration may begin before the patient issymptomatic. Any appropriate route of administration may be employed,for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols.

With respect to a subject having an elevated blood lipid level-relateddisease or disorder, an effective amount is sufficient to stabilize,slow, or reduce the progression or symptoms or presentation of theelevated blood lipid level-related disease or disorder. With respect toa subject having inflammation or a stress response, an effective amountis sufficient to stabilize, slow, reduce, or reverse the inflammation orthe stress response.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” Ed. A. R.Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

The pharmaceutical compositions may comprise any form of gallic acidthat is derived from EuMil. More specifically, the gallic acidderivative can be selected from the group comprising: methyl gallate,ethyl gallate, glycerol-1-gallate, glucose-1-gallate (GG1),glucose-6-gallate (GG6), glucose-1,6-digallate (DGG16), mucicacid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone5-gallate, geraniin, corilagin, chebilc acid, and m-digallic acid withminor p-digallic acid.

The gallic acid derivatives can be selected from a combination of gallicacid derivatives selected from the group consisting of: Compound4+Compound 5+Compound 2a, Compound 4+Compound 8+Compound 2a, Compound4+Compound 5+Compound 7, Compound 4+Compound 2a, Compound 4+Compound 2b,Compound 7+Compound 2b, Compound 5+Compound 8, and Compound 5+Compound7.

The compositions of the invention can be administered in many dosageforms. In certain examples, the dosage form is selected from a foodcomposition, tablet, pill, gel, capsule, a patch, a suspension tablet,liquid, solution, aqueous emulsion powder, lozenge, sachet, cachet,elixir, suspension, emulsion, solution, syrup, aerosol, ointment, softgelatin capsule, and hard gelatin capsule, suppository, creams, lotions,solutions, gels, pastes powder, and suspension. The food can be a healthfood product, a food product made from cereal flour, gums, a dairyproduct, a soup, a broth, a paste, a sauce, a beverage, a vitamincomplex, a food rich in cholesterol, salt, or pepper.

In certain preferred examples, the compositions of the invention can beadministered in a patch form. For example, the compounds can be adsorbedto patches which can be affixed to the skin. For example, the patchescan have use in patients with Xanthalasma, a disorder characterized byorange crystals of cholesteryl ester deposits in the eyelids.

Combination Treatments

The invention further contemplates combination therapies. In the presentinvention, the one or more gallic acid derivatives can be administeredeither alone or in combination with other agents known to affect, e.g.lipid levels, where combining compounds or extracts would lead tosynergistic effects.

In one embodiment, a composition comprising the compounds of theinvention as described herein will further comprise a therapeutic agent.

In certain examples, the therapeutic agent is selected from the groupconsisting of, but not limited to, inhibitors of cholesterol metabolism,inhibitors of triglyceride synthesis, beta blockers, diuretics,inhibitors of platelet aggregation, angiogenesis inhibitors angiogenesisinhibitors, arthritis medication, toxins, anti-inflammatory agents. Thetoxins may be, for example botulinum toxin.

The therapeutic agent may be a small molecule or macromolecule such aspeptide, protein or nucleic acid. The therapeutic agent may be selectedfrom the group consisting of bile-acid-binding resins, which interruptthe recycling of bile acids from the intestine to the liver (includingcholestyramine (QUESTRAN LIGHT, Bristol-Myers Squibb), and colestipolhydrochloride (COLESTID, Pharmacia & Upjohn Company)); statins, whichinhibit cholesterol synthesis by blocking HMG-CoA—the key enzymeinvolved in cholesterol biosynthesis (including lovastatin (MEVACOR,Merck & Co., Inc.), a natural product derived from a strain ofAspergillus, pravastatin (PRAVACHOL, Bristol-Myers Squibb Co.), andatorvastatin (LIPITOR, Warner Lambert)) cerivastatin (BAYCOR (Bayer)),fluvastatin (LESCOL (Sandoz)), and simvastatin (ZOCOR (Merck)); niacin,a water-soluble vitamin B-complex which diminishes production of VLDLand is effective at lowering LDL; fibrates, lower serum triglycerides byreducing the VLDL fraction and may in some patient populations give riseto modest reductions of plasma cholesterol via the same mechanism(including clofibrate (ATROMID-S, Wyeth-Ayerst Laboratories), andgemfibrozil (LOPID, Parke-Davis)]; estrogen replacement therapy, lowerscholesterol levels in post-menopausal women; long chain alpha,omego-dicarboxylic acids have been reported to lower serum triglycerideand cholesterol (See, e.g., Bisgaier et al., 1998, J. Lipid Res.39:17-30; WO 98/30530; U.S. Pat. No. 4,689,344; WO 99/00116; U.S. Pat.Nos. 5,756,344; 3,773,946; 4,689,344; 4,689,344; 4,689,344; and3,930,024); other compounds including ethers (See, e.g., U.S. Pat. Nos.4,711,896; 5,756,544; 6,506,799), phosphates of dolichol (U.S. Pat. No.4,613,593), and azolidinedione derivatives (U.S. Pat. No. 4,287,200) aredisclosed as lowering serum triglyceride and cholesterol levels.

Preferably, the therapeutic agent is attached to the gallic acidderivative by a covalent linkage. Any covalent linkage is appropriatefor attachment of the gallic acid derivative and the therapeutic agent.

The therapeutic agent can be administered in combination with the one ormore gallic acid derivatives.

In certain embodiments, the compounds of the invention can beadministered with other agents to achieve a therapeutic effect, e.g. canbe administered with other therapeutics to treat an elevated blood lipidlevel-related disease.

In some embodiments, the amount of compound, e.g. any one of thecompounds 2a-13 as described herein, that is required to achieve atherapeutic effect, when co-administered with another agent that has aneffect to treat an elevated blood lipid level-related disease, is lessthan about 85% of the amount of compound (e.g. compound 2a-13 orcombinations thereof) required to achieve the therapeutically effectwhen administered in the absence of the other therapeutic (e.g., lessthan about 80%, less than about 75%, less than about 70%, less thanabout 65%, less than about 60%, less than about 55%, or less than about50%). In certain examples, the invention contemplates the administrationof other therapeutic agents which possess the capacity to reduce thelevel of cholesterol and/or LDL and/or triglycerides and/or other lipidsin the blood of a mammal, and are considered useful in a composition incombination with compounds of the present invention.

In one embodiment, one or more of the compounds of the instant inventionand one or more agents, e.g. a therapeutic agent, are administeredsimultaneously in a combined amount effective to produce the desiredtherapeutic outcome. This is achieved by administering a singlecomposition or pharmacological formulation that includes all of theactive agents, or by administering to the subject two distinctcompositions or formulations, at the same time, wherein one compositionincludes one or more of the compounds of the instant invention, and thesecond composition includes the therapeutic agent. The one or more ofthe compounds of the instant invention and other therapeutic agent(s)may or may not exhibit the same mechanism by which they reduce thelevels of total cholesterol (i.e., hypercholesteremia) and/or otherlipids in a mammal.

Alternatively, treatment with the one or more of the compounds of theinstant invention may precede or follow therapy with another therapeuticagent by intervals ranging from minutes to weeks. In embodiments wheretwo or more therapeutic compositions are administered separately, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that the second therapeuticagent and one or more of the compounds of the instant invention wouldstill be able to exert an advantageously combined effect. In suchinstances, it is contemplated that one would administer one or morecompositions within about 12-24 hours of each other, or about 6-12 hoursof each other, or with a delay time of only about 12 hours. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Exemplary therapeutic agents that can be used in combination therapyinclude, but are not limited to, the following:

Statin-Related Agents

In one embodiment, the therapeutic agent in the combination therapy is astatin-related agent. The term “statin-related” refers to any statindrug that is presently on the market, or is modified from the presentlymarketed statin drugs, and has a therapeutic effect when combined withthe compositions described herein. As such it should be understood thatanalogs and variants of preexisting statins are contemplated to beuseful herein. Such analogs or variants may be produced through rationaldrug design techniques known to those of skill in the art. Inparticular, statin drugs are known as HMG-CoA reductase inhibitors.These drugs are presently in clinical use as drugs in the battle againsthigh cholesterol and in the control of heart attacks, both recurrent andfirst heart attacks. These agents generally have few side effects, andhelp not only to lower overall cholesterol, LDL cholesterol andtriglycerides, but also to increase HDL cholesterol. The use of othercompounds in the combination therapy that interfere with the activity ofHMG-CoA reductase is considered as an aspect of the invention.

Statins are exemplified by lovastatin (CAS Registry No. 75330-75-5; alsoknown as mevinolin or monacolin K), and analogs of this compound havebeen described in numerous publications and patents. Exemplary statincompositions that are commercially available include LIPITOR(atorvastatin), PRAVACHOL (pravastatin), ZOCOR (simvastatin), MEVACOR(lovastatin), and LESCOL (fluvastatin). Methods of preparing suchcompounds are well known to those of skill in the art (see e.g., U.S.Pat. Nos. 6,521,762; 4,420,491; 4,342,767; 4,319,039; 4,294,846;4,444,784; 4,582,915 and 4,820,850, all of which are incorporated byreference in their entireties herein). As described in the foregoingpatents, statins are traditionally produced through fermentation usingorganisms from the Aspergillus genus, Monascus genus, Pleurotus genus,Coniothyrium genus and the like (see U.S. Pat. No. 6,521,762,incorporated by reference herein for review of fermentation procedures).

Moreover, formulations of statins as pharmaceutical medicament have beendescribed in e.g., the Physician's Desk Reference. For example, tabletformulations of LIPITOR (atorvastatin calcium) are described at pages2547-2551 (Parke-Davis, NJ.) and 2610-2613 (Pfizer, NY) of thePhysician's Desk Reference (57.sup.th Edition, 2003). These formulationsare supplied as tablets of atorvastatin calcium containing 10 mg, 20 mg,40 mg, 50 mg, and 80 mg atorvastatin. The tablets are administered indoses ranging from 10 mg/day to 80 mg/day. The compositions of LIPIORpresently being used to lower cholesterol in humans may be used in thecombined treatments of the present invention to produce a therapeuticamelioration of elevated lipid related diseases or disorders.

PRAVACHOL (pravastatin sodium; Bristol-Myers Squibb, NY), is anotherexemplary commercially available statin that may be used in the combinedtherapies of the present invention. PRAVACHOL is supplied as a 10 mg, 20mg, 40 mg, and 80 mg tablets. These tablets may be administered at adaily dose of ranging from 10 mg/day to 80 mg/day. In exemplarytreatments for hypercholesterolemia, 40 mg/day are administered as asingle daily dose, with or without food. However, it is generallyappreciated that this dose may be increased or lowered depending on thelevel of renal and liver function of the patient being treated. Theadministration doses and treatment guidelines for PRAVACHOL arediscussed in further detail at pages 1101-1105 of the Physician's DeskReference (57.sup.th Edition, 2003) and may be used to provide guidancefor the use of statins in the methods of the present invention.

ZOCOR (simvastatin; Merck & Co., Inc., NJ), is another exemplary statincomposition that may be used in combination with the layeredphyllosilicate material of the present invention. Formulations ofsimvastatin are described at pages 2126-2131 of the Physician's DeskReference (57.sup.th Edition, 2003). The daily doses may range from 5mg/day to 80 mg/day and those of skill in the art are referred to thePhysician's Desk Reference for further guidance regarding treatmentprotocols that may be used and/or modified for the present invention.

MEVACOR (lovastatin; Merck & Co., Inc. NY), and LESCOL (fluvastatin) areother exemplary statins that are described in the Physician's DeskReference (57th Edition, 2003) at pages 2036-2041 and 2283-2287,respectively. Those of skill in the art will readily be able to modifythe above-referenced pharmaceutical compositions that comprise variousstatin-related agents for the methods of the present invention.

For treatment protocols, those of skill may use the guidelines used forthe any of the above-referenced pharmaceutical statins. Administrationof ordinary tablets containing statin once, twice, three or more times aday. Accordingly, the skilled artisan may use dosages that havepreviously proven effective for the above indications as a preliminarymeasure of the amount of any of the above-referenced statins, to use inthe therapeutic methods of the invention. Oral doses of the statins areparticularly contemplated. Such oral doses may comprise theadministration of between about 5 mg to about 80 mg statin drug on adaily basis. However, larger doses e.g., up to 200 mg/day also may beused. It should be understood the subject may receive more or less ofthe statin or other cholesterol lowering agent. Also it should beunderstood that similar doses may be administered through other routineroutes of administration. The statin or other cholesterol lowering agentmay be delivered in a single dose or alternatively may be subdivided andadministered in multiple doses over a given period of time.

Nicotinic Acid

In another embodiment, the therapeutic agent in the combination therapyis nicotinic acid. Nicotinic acid (niacin) lowers total and LDLcholesterol and raises HDL cholesterol, and also lowers triglycerides.The dose of niacin required to lower cholesterol is about 100 times morethan the Recommended Daily Allowance (RDA) for niacin and thus canpotentially be toxic. Therefore, the drug must be taken under a doctor'scare.

Fibrates

In yet another embodiment, the therapeutic agent in the combinationtherapy is a fibrate. Fibric acid derivatives (fibrates) are a class ofmedications that lower blood triglyceride levels. Fibrates lower bloodtriglyceride levels by reducing the liver's production of VLDL and byspeeding up the removal of triglycerides from the blood. Fibrates arealso modestly effective in increasing blood HDL cholesterol levels;however, fibrates are not effective in lowering LDL cholesterol.Exemplary fibrates include, but are not limited to, Bezafibrate (e.g.BEZALIP), Ciprofibrate (e.g. MODALIM), Clofibrate, Gemfibrozil (e.g.LOPID) and Fenofibrate (e.g. TRICOR).

Bile Acid Resins

In still another embodiment, the therapeutic agent in the combinationtherapy is a bile acid resin. Bile acid resins, also known as bile acidsequesterants, are mainly used to treat patients with moderatelyelevated LDL-cholesterol and when cholesterol-lowering drug therapy isnecessary in young adult men and premenopausal women. They are alsosometimes combined with other cholesterol-lowering drugs like “statins”to decrease very high levels of cholesterol. Exemplary bile acid resinsinclude, but are not limited to, Cholestyramine, Colestipol (Colestid),and Cholsevelam (Welchol).

Cholesterol Absorption Inhibitors

In yet another embodiment, the therapeutic agent in the combinationtherapy is a cholesterol absorption inhibitor. Ezetimibe (ZETIA) is theonly prescription drug currently in this class. This drug preventsdietary cholesterol from being absorbed from the small intestine andentering the blood, thus lowering blood cholesterol levels. Synergisticcompositions comprising a cholesterol absorption inhibitor and thelayered phyllosilicate material is particularly contemplated.

Salicylic Acid

Also contemplated as a therapeutic agent in the combination therapy issalicylic acid (aspirin). Aspirin has been shown to have a protectiveeffect against heart attacks in patients with clogged blood vessels, andcan also be used in a composition according to the present invention.The cholesterol-reducing mechanism is believed to be based on the acidicproperties of aspirin, and as such the acid deconjugates thebile:cholesterol complex, reducing bioavailability.

Phytosterols

In another embodiment, the therapeutic agent in the combination therapyis a phytosterol. Phytosterols, also known as plant sterols or stanols,are lipids having chemical structures similar to cholesterol, which arepresent in all plants including but not limited to vegetables, fruits,and grains, particularly in nuts, seeds, and plant oils. Phytosterolsinhibit intestinal cholesterol absorption, thereby lowering plasma totaland low-density lipoprotein (LDL) cholesterol levels. Daily consumptionof about one to two grams of phytosterols reduces the risk forcardiovascular disease by about 25 to about 28% without causing anyadverse effects. Twice per day consumption of about 0.40 grams ofphytosterols or about 0.65 grams of phytosterol esters has also beenshown to lower total cholesterol levels and LDL cholesterol levels by upto 10%. An extract of the soy plant—sitosterol—is available in a productcalled TAKE CONTROL (Lipton). And an extract of pineneedles—sitostanol—is available in a similar product called BENECHOL(McNeil). The use of policosanol, derived from waxes of various plantsincluding, but not limited to, sugar cane and yams, is alsocontemplated.

Alginates and Pectins

In yet another embodiment, the therapeutic agent in the combinationtherapy is a polysaccharide including but not limited to, alginate,pectin, gellan gum, xanthan gum and zooglan. Alginates, pectins andmodifications thereof are reported to interact with dietary cholesteroland affect its absorption and excretion (see U.S. Pat. Nos. 5,141,927;5,597,810; 7,026,302, Japanese Patent No. 09235234, and Kimura et al.,J. Ethnopharmacol, 54(1):47 54 (1996), the disclosures of which areincorporated herein by reference in their entireties).

Lecithin

In another embodiment, the therapeutic agent in the combination therapyis Lecithin (CAS# 8002-43-5). Leithin is usually used as a synonym forphosphatidylcholine, a phospholipid which is the major component of aphosphatide fraction isolated from egg yolk or soy beans. Lecithin iscommercially available in high purity as a food supplement and formedical uses. For example, Lecithin 19 Grain is sold over the counterand claims that it reduces cholesterol.

In yet another embodiment, the compounds of the invention can beutilized as delivery vehicles. In one variation, the compounds can beused to deliver nucleic acids or proteins, for example an antibodyagainst cholesterol. In other embodiments, gene constructs for SREBP-1(sterol regulatory element binding transcription factor 1) or LDLreceptor may be used. In another variation, the compound or compositionis a delivery vehicle for a therapeutic agent described herein. Bindingof a therapeutic agent to a compound of the invention can improve itsdelivery and subsequent absorption through mucosal membranes, includingthe ocular, dermal, nasal and intestinal membranes. Drug release fromthe compound can be induced by pH, ionic strength changes, and/or inresponse to temperature, ionic current or ultrasound.

In other embodiments, the compounds of the invention are used in lieu ofor in conjunction with other drug delivery vehicles known in the art inorder to increase cell targeting membrane permeability and absorption.Exemplary drug delivery systems known in the art include, but are notlimited to, those described in U.S. Pat. Nos. 6,838,528; 6,797,704;6,730,334; 6,706,289; 6,482,439; 6,443,989; 6,383,478; 6,165,440;5,780,044; 5,759,563; 5,565,215; and U.S. Patent Application PublicationNos. 2007/0059327; 2007/0053845; 2007/00036278; 2007/0031340;2007/0026048; 2007/0003610; 2006/0193787; 2006/0188543; 2006/0149392;2006/0105049; 2006/0057206; 2006/0034925; 2005/0266090; 2005/0260276;2005/0249798; 2005/0249774; 2005/0220754; 2005/0209345; 2005/0058603;2005/0152965; 2005/0089572; 2005/0058701, the disclosures of which areall incorporated herein by reference in their entireties.

In one embodiment, the mammal is human. In other embodiments, the mammalis an animal. Exemplary animals include, but are not limited to, farmanimals such as horses, cows, sheep, pigs, alpacas, llamas, camels,birds, and goats; companion animals such as dogs and cats; exotic and/orzoo animals; laboratory animals including mice, rats, rabbits, guineapigs and hamsters.

VI. Nutraceuticals

The compositions of the invention can be formulated as nutraceuticals.

In certain embodiments, gallic acid derivatives can be used alone. Forexample, a EuMil extract can be used alone.

In certain examples, a nutraceutical is any substance that is a food ora part of a food or something that is added to a food, and providesmedical or health benefits, including the prevention and/ or thetreatment of a disease or disorder. Nutraceuticals are often naturalproducts that are identified from botanicals, and purified or partiallypurified and then included in foods.

The nutraceutical can be administered as a food, a food composition, atablet, pill, gel, capsule, suspension tablet, liquid, solution, aqueousemulsion powder, lozenge, sachet, cachet, elixir, suspension, emulsion,solution, syrup, aerosol, ointment, soft gelatin capsule, and hardgelatin capsule, suppository, creams, lotions, solutions, gels, pastespowder, and suspension. The compounds, in certain preferred embodiments,may be adsorbed to patches and adhered to the skin surface.

The food can be a health food product, a food product made from cerealflour, gums, a dairy product, a soup, a broth, a paste, a sauce, abeverage, a vitamin complex, a food rich in cholesterol, salt, orpepper.

Gallic acid derivatives of the present invention can be used as abeverage. For example, the beverage can be a beverage that containswater, a sweetener and a suitable additive.

The nutraceuticals can be made as a dietary supplement. For dietarysupplements, the extract can be added and mixed according to methodsroutine in the art. Dietary supplements for animals can be prepared in avariety of forms including, but not limited to, liquid, powder, or solidpill forms. In the present invention, the one or more gallic acidderivatives can be administered either alone or in combination withother agents known to affect, e.g. lipid levels, where combiningcompounds or extracts would lead to synergistic effects or additiveeffects.

Nutraceutical compositions can be packaged in a number of ways.

The controlled release of a physiologically active agent into anindividual's system from a sustained release dosage form is often adesirable alternative to repeated administrations (e.g., after aprescribed number of hours) of the agent from conventional dosage forms(i.e., one controlled release dosage may provide the full daily regimenof active agent). Sustained release formulations incorporating watersoluble cellulose ethers in a hydrophilic solid core matrix have beendeveloped for this purpose. Upon contact with an aqueous environment,such formulations hydrate to form a gel layer on the surface of thesolid core matrix, which limits entry of water into the solid core,thereby establishing a diffusion-controlled sustained release of theactive ingredient therein.

Alternative sustained release formulations are based on solid corematrixes that have been coated with materials that can form membranesexhibiting sustained release characteristics. Examples of such materialsinclude hydroxypropyl methylcellulose, shellac, fats, and waxes. Onepreparation for the sustained release of a nutraceutical includes asolid matrix coated with a coating comprising methylcellulose. The solidmatrix contains a polyphenol and a therapeutically effective amount of anutraceutical. U.S. Pat. No. 7,115,283, incorporated by reference in itsentirety herein, describes preparations and methods for the controlledrelease of nutraceuticals. In certain examples, a biodegradable matrixmaterial can be used for the time dependent release of compounds e.g.used in drug eluting stents to reach small capillaries in the brain.

U.S. Pat. No. 6,723,358, incorporated by reference in its entiretyherein, describes an edible matrix composition that has a chewabletexture and that contains at least one encapsulated component.Encapsulation of food components is described in Encapsulation andControlled Release of Food Ingredients, edited by S. J. Risch and G. A.Reineccius, ACS Symposium Series 590 (1995). U.S. Pat. No. 5,183,690 toCarr, et. al. describes a continuous extrusion process usingstarch-based material to encapsulate components. The resulting productsare in the form of particulates, which have gelatinized starch as acontinuous domain, in which discontinuous domains of biologically activecore material is entrapped.

VII. Dosage and Adminisration

The compositions of the invention alone, or in combination with one ormore therapeutic agents as described herein, is administered by anyroute that delivers an effective dosage to the desired site of action,with acceptable (preferably minimal) side-effects. Numerous routes ofadministration are known, including for example, oral, rectal, vaginal,transmucosal, buccal or intestinal administration; parenteral delivery,including intraperitoneal intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, cutaneous or intradermal injections;respiratory or inhalation, nasal, pulmonary and topical application,including ocular and transdermal applications.

When used in the above or other treatments, a “therapeutically effectiveamount” or an “effective amount” of a compound or a compositioncomprising a compound of the invention means a sufficient amount of oneor more of the compounds of the invention is provided to treatdisorders, at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood, however, that the total daily usage ofthe one or more compounds will be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the compound at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

The total daily dose of compositions of the invention administered to amammalian subject range from about 10 to about 500 mg/kg/day. Inpreferred embodiments, when a single compound is administered, the dosegiven is preferably in the range of 40 to 200 mg/kg/ day. If desired,the effective daily dose may be divided into multiple doses for purposesof administration; consequently, single dose compositions may containsuch amounts or submultiples thereof to make up the daily dose. Thedosage regimen of a composition of the invention alone or in combinationas described herein to be used in anti-cholesterol treatment will bedetermined by the attending physician considering various factors whichmodify the action of the administered composition, e.g., the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors.

Multiple doses per day are contemplated to achieve appropriate systemiclevels of the compositions of the present invention.

Since Applicants have shown that 40 mg daily intake of Simvastatin canlower blood levels of cholesterol, a range of 40 mg-200 mg per day of asingle compound, for example in preferred embodiments compound 4, givenalone, should be as effective as or better than simvastatin in loweringcholesterol, in increasing cellular efflux of cholesterol or inhibitingcellular uptake of cholesterol.

Likewise Vytorin (a mixture of 40 mg of Simvastatin+10mg of ezetimibe)can lower blood cholesterol to 50%. In certain embodiments, differentdosages of the compounds can be combined, for example one dosage levelof a cholesterol lowering gallate derivative (e.g. compound 8) withanother dosage level of a cholesterol uptake/absorption inhibitor (e.g.compound 3).

In preferred examples, the dosage comprises about 0.1% to about 95%gallic acid derivative weight to weight of the composition, for example0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 70%, 75%,80%, 85%, 90%, 95% or more.

Oral dosage forms include tablets, capsules, caplets, solutions,suspensions and/or syrups, and may also comprise a plurality ofgranules, beads, powders or pellets that may or may not be encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts, e.g., in Remington: The Science and Practice ofPharmacy, supra). Tablets and capsules represent the most convenientoral dosage forms, in which case solid pharmaceutical carriers areemployed.

Tablets may be manufactured using standard tablet processing proceduresand equipment. One method for forming tablets is by direct compressionof a powdered, crystalline or granular composition containing the activeagent(s), alone or in combination with one or more carriers, additives,or the like. As an alternative to direct compression, tablets can beprepared using wet-granulation or dry-granulation processes. Tablets mayalso be molded rather than compressed, starting with a moist orotherwise tractable material.

In addition to the compositions of the invention alone, or incombination as described herein, tablets prepared for oraladministration will generally contain other materials such as binders,diluents, lubricants, disintegrants, fillers, stabilizers, surfactants,preservatives, coloring agents, flavoring agents and the like. Bindersare used to impart cohesive qualities to a tablet, and thus ensure thatthe tablet remains intact after compression. Suitable binder materialsinclude, but are not limited to, starch (including corn starch andpregelatinized starch), gelatin, sugars (including sucrose, glucose,dextrose and lactose), polyethylene glycol, propylene glycol, waxes, andnatural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose, ethylcellulose, hydroxyethyl cellulose, and the like), and Veegum. Diluentsare typically necessary to increase bulk so that a practical size tabletis ultimately provided. Suitable diluents include dicalcium phosphate,calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride,dry starch and powdered sugar. Lubricants are used to facilitate tabletmanufacture; examples of suitable lubricants include, for example,vegetable oils such as peanut oil, cottonseed oil, sesame oil, oliveoil, corn oil, and oil of theobroma, glycerin, magnesium stearate,calcium stearate, and stearic acid. Disintegrants are used to facilitatedisintegration of the tablet, and are generally starches, clays,celluloses, algins, gums or crosslinked polymers. Fillers include, forexample, materials such as silicon dioxide, titanium dioxide, alumina,talc, kaolin, powdered cellulose and microcrystalline cellulose, as wellas soluble materials such as mannitol, urea, sucrose, lactose, dextrose,sodium chloride and sorbitol. Stabilizers are used to inhibit or retarddrug decomposition reactions that include, by way of example, oxidativereactions. Surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents.

The dosage form may also be a capsule, in which case the materialcontaining the compositions of the invention may be encapsulated in theform of a liquid or solid (including particulates such as granules,beads, powders or pellets). Suitable capsules may be either hard orsoft, and are generally made of gelatin, starch, or a cellulosicmaterial, with gelatin capsules preferred. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.(See, for e.g., Remington: The Science and Practice of Pharmacy, supra),which describes materials and methods for preparing encapsulatedpharmaceuticals.

Solid dosage forms, whether tablets, capsules, caplets, or particulates,may, if desired, be coated so as to provide for delayed release. Dosageforms with delayed release coatings may be manufactured using standardcoating procedures and equipment. Such procedures are known to thoseskilled in the art and described in the pertinent texts (See, for e.g.,Remington: The Science and Practice of Pharmacy, supra). Generally,after preparation of the solid dosage form, a delayed release coatingcomposition is applied using a coating pan, an airless spray technique,fluidized bed coating equipment, or the like. Delayed release coatingcompositions comprise a polymeric material, e.g., cellulose butyratephthalate, cellulose hydrogen phthalate, cellulose proprionatephthalate, polyvinyl acetate phthalate, cellulose acetate phthalate,cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulosesuccinate, carboxymethyl ethylcellulose, hydroxypropyl methylcelluloseacetate succinate, polymers and copolymers formed from acrylic acid,methacrylic acid, and/or esters thereof.

Created as an alternate route of drug administration to improve patientcompliance and reduce drug side effects, prescription skin patches arerapidly becoming an important healthcare product category. Advances insynthetic materials and patch design have led to patches that are moreesthetically acceptable and that are capable of delivering sustaineddosing of active compounds for several days in a smaller package.

Patches are advantageous for long term treatment. Long-term treatment ispossible for those who require longer periods of treatment than areprovided for by conventional treatment methods. For example, the presentinvention provides for a pre-determined period of treatment which maylast as long as, for example, up to 1, 2, or more years after treatmentbegins.

The dosage form can be an immediate release, sustained release, anddelayed release.

Sustained release dosage forms provide for drug release over an extendedtime period, and may or may not be delayed release. Generally, as willbe appreciated by those of ordinary skill in the art, sustained releasedosage forms are formulated by dispersing a drug within a matrix of agradually bioerodible (hydrolyzable) material such as an insolubleplastic, a hydrophilic polymer, or a fatty compound, or by coating asolid, drug-containing dosage form with such a material. Insolubleplastic matrices may be comprised of, for example, polyvinyl chloride orpolyethylene. Hydrophilic polymers useful for providing a sustainedrelease coating or matrix cellulosic polymers include, withoutlimitation: cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetatephthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulosephthalate, hydroxypropylcellulose phthalate, cellulosehexahydrophthalate, cellulose acetate hexahydrophthalate, andcarboxymethylcellulose sodium; acrylic acid polymers and copolymers,preferably formed from acrylic acid, methacrylic acid, acrylic acidalkyl esters, methacrylic acid alkyl esters, and the like, e.g.copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethylacrylate, methyl methacrylate and/or ethyl methacrylate, with aterpolymer of ethyl acrylate, methyl methacrylate andtrimethylammonioethyl methacrylate chloride (sold under the tradenameEudragit RS) preferred; vinyl polymers and copolymers such as polyvinylpyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetatecrotonic acid copolymer, and ethylene-vinyl acetate copolymers; zein;and shellac, ammoniated shellac, shellac-acetyl alcohol, and shellacn-butyl stearate. Fatty compounds for use as a sustained release matrixmaterial include, but are not limited to, waxes generally (e.g.,carnauba wax) and glyceryl tristearate.

Although the present compositions may be administered orally, othermodes of administration are contemplated as well. Exemplary modes ofadministration include transmucosal (e.g., U.S. Pat. Nos. 5,288,498;6,248,760; 6,355,248; 6,548,490, the disclosures of which areincorporated herein by reference in their entireties), transurethral(e.g., e.g., U.S. Pat. Nos. 5,919,474 and 5,925,629, the disclosures ofwhich are incorporated herein by reference in their entireties), vaginalor perivaginal (e.g., U.S. Pat. Nos. 4,211,679; 5,491,171 and 6,576,250,the disclosures of which are incorporated herein by reference in theirentireties) and intranasal or inhalation (e.g., U.S. Pat. Nos.4,800,878; 5,112,804; 5,179,079; 6,017,963; 6,391,318 and 6,815,424, thedisclosures of which are incorporated herein by reference in theirentireties). One of skill in the art would be able to modify acomposition as described herein alone or in combination to be used inany of the modes of administration described herein.

The compositions of this invention can be employed in mixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for topical application which donot deleteriously react with the acid or the alcohol in the composition.The compositions of the invention can also include diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980).

VIII. Kits

In related variations of the preceding embodiments, a compositioncomprising the compounds of the invention alone or in combination asdescribed herein may be so arranged, e.g., in a kit or package or unitdose, to permit co-administration with one or more other therapeuticagents. In another aspect, the one or more gallic acid derivatives andthe agent are in admixture. In some embodiments, the two components tothe kit/unit dose are packaged with instructions for administering thetwo agents to a mammalian subject for treatment of one of theabove-indicated disorders and diseases.

The kits can comprise any of the pharmaceutical compositions asdescribed herein, and instructions for use.

In certain preferred examples, the invention features a kit for the usein preventing or treating an elevated blood lipid level-related diseasein a mammal comprising one or more gallic acid derivatives.

In other examples, the invention features kits that can be used inpreventing or treating inflammation or an elevated stress response.

In other examples, the invention features kits that can be use inreducing cholesterol biosynthesis, increasing the cellular efflux ofcholesterol, or inhibiting the cellular uptake of cholesterol.

The one or more gallic acid derivatives can be selected from the groupcomprising: methyl gallate, ethyl gallate, glycerol-1-gallate,glucose-1-gallate (GG1), glucose-6-gallate (GG6), glucose-1,6-digallate(DGG16), mucic acid-2-gallate, 1-methyl mucate-2-gallate, mucic acid1,4-lactone 5-gallate, geraniin, corilagin, chebilc acid, and m-digallicacid with minor p-digallic acid, and instructions for use.

Additionally, the gallic acid derivatives in the kits can beadministered as a nutraceutical.

In certain embodiments, the kits comprise: Compound 4+Compound5+Compound 2a, Compound 4+Compound 8+Compound 2a, Compound 4+Compound5+Compound 7, Compound 4+Compound 2a, Compound 4+Compound 2b, Compound7+Compound 2b, Compound 5+Compound 8, and Compound 5+Compound 7.

The following examples are offered by way of illustration, not by way oflimitation. While specific examples have been provided, the abovedescription is illustrative and not restrictive. Any one or more of thefeatures of the previously described embodiments can be combined in anymanner with one or more features of any other embodiments in the presentinvention. Furthermore, many variations of the invention will becomeapparent to those skilled in the art upon review of the specification.The scope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

EXAMPLES Example 1 EuMil Mixture and Identification of IndividualComponents

Ayurvedic studies have suggested that several herbal mixtures exerttheir effect in a synergistic manner Moreover, since EuMil is made up ofa mixture of EO, W. somnifera and O. sanctum (with Asparagus racemosusused as a filler for pill preparation), the first study focused onwhether this herbal preparation exerted its hypocholesterolemic effectin a synergistic manner or whether the activity was confined to only oneof the above herbal ingredients.

The cell-based bioassays described herein revealed that only the EOcomponent of EuMil significantly inhibited de novo biosynthesis ofcholesterol. Bioassay-guided isolation from an EO aqueous alcoholicsolution led to the isolation of purified gallic acid and mucic acidderivatives 1-9 as identified in Table 1, shown below. The structures ofcompounds 1-9 are shown in FIG. 1. These compounds are shown to have thehypocholesterolemic activity of the EO extract as demonstrated in theExamples described herein. In addition, compounds 10 through 13 of Table1 were also isolated and their structures are shown in FIG. 1. Compounds10-13 are also tested in the cell-based bioassay.

TABLE 1 FIG. NO. COMPOUND NAME REFERENCE NO. 1 gallic acid  2a methylgallate  2b ethyl gallate 3 glycerol-1-gallate 17-19 4 glucose-1-gallate(GG1) 20   5 glucose-6-gallate (GG6) 17, 19 6 glucose-1,6-digallate 17,19, 21 (DGG16) 7 mucic acid-2-gallate 20, 22 8 1-methyl mucate-2-gallate20.22 9 mucic acid 1,4-lactone 5- 20, 22 gallate 10  geraniin 11 corilagin 12  chebulic acid 13  m-digallic acid along with minorp-digallic acid

The structure of each of these purified compounds isolated from thefruit of EO is shown in FIG. 1.

The structures of compounds 1 to 13 were determined by mass spectroscopy(MS) and nuclear magnetic resonance (NMR) spectroscopy, as well ascomparison with previously reported data [17-22]. The 1H NMR spectrum of(GG6) (5) displayed resonances for both the α- and β-anomers, as wasexpected as all of these compounds have been previously isolated from EOexcept for glycerol-1-gallate (3) and GG6 (5).

Extraction and Polyamide Treatment of the Plant Components of EuMil

Plant powder from each component of EuMil was extracted with methanol. Aportion of the methanol extract of each plant was eluted throughPolyamide SC 6 (0.05-0.16 mm, Macherey-Nagel) to remove polymerictannins and tested in the cell-based assays. Only the plant powder fromEO was active, which indicated that most, if not all of thehypocholesterolemic properties of EuMil reside with the EO component ofEuMil.

Crude EO powder (500 mg) was extracted with 25% ethanol in water (2×10ml) and the extract dried by rotary evaporation to provide a crudeextract (240 mg). The crude extract was dissolved in water (4 ml),centrifuged and the supernatant separated using the preparativereversed-phase HPLC (see FIG. A1 see Appendix) (4 injections of 60 mg,YMC pack ODS-AQ column, RP18, 150×20 mm, 5 μM, isocratic in 100% H₂O(with 0.1% HCOOH) for 30 minutes and linear gradient from 0% to 30%acetonitrile (with 0.1% HCOOH) for 40 minutes, flow rate 12 mL/min, UVdetection 210 and 254 nm) to obtain mucic acid-2-gallate (7) (19.6 mg),mucic acid 1,4-lactone 5-gallate (9) (16.0 mg), 1-methylmucate-2-gallate (8) (3.6 mg), a mixture of gallic acid (1) andglucose-1-gallate (GG1) (4) (9 mg mixture), glucose-6-gallate (GG6) (5)(1.2 mg), glucose-1,6-digallate (DGG16) (6) (5.2 mg) andglycerol-1-gallate (3) (2.4 mg) in order of elution (structures in FIG.1). Methyl gallate (2a) and ethyl gallate (2b) were present in theextract when the percentage of methanol or ethanol respectivelyapproached 50% and above and could be isolated using the samepreparative HPLC conditions. Each of the compounds were characterized by(−)-HR-ESI MS and 1H, COSY, HSQC and HMBC NMR experiments and theirspectroscopic data compared to previously reported data. The retentiontime of each compound was obtained using aYMC Pack-ODS-AQ column (S-5mm, 150×4.6 mm, flow rate 1.0 mL/min) using isocratic 100% H2O (with0.1% HCOOH) for 5 minutes and linear gradient from 0% to 30%acetonitrile (with 0.1% HCOOH) for 30 minutes.

The following purified compounds were identified and analyzed by the 25%ethanolic extraction:

Gallic acid (1): white powder; Rt 7.2 min; ESI-MS [M−H]—169.0136; 1H NMR(D2O) d 7.14 (2H, s, galloyl). Identical to gallic acid purchased fromSigma Aldrich.

Methyl gallate (2a): white powder; Rt 14.9 min; ESIMS [M−H]—183.0294; 1HNMR (D2O) d 3.83, (3H, s, OCH3), 7.14 (2H, s, galloyl).

Ethyl gallate (2b): white powder; Rt 19.0 min; ESIMS [M−H]—197.0446; 1HNMR (DMSO-d6), d 1.26 (t, J=7.6 Hz, OCH2CH3), 4.18 (q, J=7.6 Hz,OCH2CH3), 6.92 (2H, s, galloyl).

Glycerol-1-gallate (3): colourless oil, Rt. 13.2 min.; ESIMS[M−H]—243.0457; 1H NMR (D2O) d 3.67, (1H, dd, J=12.0, 6.6 Hz, H-3),3.73, (1H, dd, J=12.0, 4.7 Hz, H-3), 4.08 (1H, m, H-2), 4.34 (1H, dd,J=11.7, 6.0 Hz, H-1), 4.37 (1H, dd, J=11.7, 3.8 Hz, H-1), 7.19 (2H, s,galloyl).

Glucose-1-gallate (GG1) (4): white powder; Rt. 7.46 min; ESIMS(M−H)—331.0665; 1H NMR (D2O) d 3.50 (1H, dd, J=9.4, 9.4 Hz, H-4), 3.65(3H, m, H-2, H-3 and H-5), 3.75 (1H, dd, J=12.6, 5.6 Hz, H-6b), 3.91(1H, dd, J=12.6, 2.2 Hz, H-6a), 5.74 (1H, d, 7.8 Hz, H-1), 7.23 (2H, s,galloyl).

Glucose-6-gallate (GG6) (5): white powder; Rt 16 4 min; ESIMS[M−H]—331.0678; 1H NMR (D2O) d 3.28-4.58 (H-2 to H-5 corresponding tothe α and β isomers), 4.7 (1H, under residual solvent peak, H-1β), 5.23(1H, d, J=4.1 Hz, H-1α), 7.18 (2H, s, galloyl).

Glucose-1,6-digallate (DGG16) (6): white powder; Rt 18.2 min, ESIMS[M−H]—483.0778; 1H NMR (DMSO-d6) d 3.30 (3H, m) and 3.59 (1H, m, H-2,H-3, H-4, H-5), 4.22 (1H, dd, J=12.0, 5.0 Hz, H-6b), 4.39 (1-H, dd,J=12.0, 1.9 Hz, H-6a), 5.55 (1H, d, J=8.2 Hz, H-1), 6.99 (2H, s,galloyl), 6.93 (2H, s, galloyl).

Mucic acid-2-gallate (7): colourless paste; Rt 2.7 min; ESIMS[M−H]—361.0402; 1H NMR (D2O) d 4.15 (1H, d, J=10.1, H-4), 4.39 (1H, dd,J=10.1, 1.3 Hz, H-3), 4.62 (1H, s, H-5), 5.51 (1H, d, J=1.3 Hz, H-2),7.27 (2H, s, galloyl).

1-Methyl mucate-2-gallate (8): colourless paste; Rt 10.5 min; ESIMS[M−H]—375.0551; 1H NMR (D2O) d 3.84 (3H, s, OCH3), 4.12 (1H, dd, J=10.1,1.6 Hz, H-4), 4.36 (1H, dd, J=10.1, 2.2 Hz, H-3), 4.47 (1H, d, J=1.9 Hz,H-5), 5.56 (1H, d, J=1.9 Hz, H-2), 7.28 (2H, s galloyl).

Mucic acid 1,4-lactone 5-gallate (9): colourless paste; Rt 4.9 min;ESIMS [M−H]—343.0295; 1H NMR (D2O) d d4.41 (1H, dd, J=8.8, 8.7 Hz, H-3),4.8 (1H, under residual solvent peak, H-2), 4.90 (1H, dd, J=8.5, 2 Hz,H-4), 5.48 (1H, s, H-5), 7.08 (2H, s galloyl).

1H, 13C and 2D NMR spectra were acquired on a BRUKER 500 MHz instrumentusing standard pulse sequences and parameters. NMR spectra werereferenced to the residual protons in DMSO-d6 at δ2.49 or in D2O withacetonitrile (δ2.06) as an internal reference. Preparative HPLC wasperformed on a Gilson HPLC with a Gilson 322 pump, Gilson UV/VIS-156,with a Gilson 215 liquid handler using the UNIPOINT software. All of thecompounds were analyzed by analytical HPLC using an Agilent 1100 HPLCsystem with a G1311A quaternary pump, G1313A ALS, G1315DAD, G1316ACOLCOM and a G1322A degasser. High resolution electrospray mass spectraldata were recorded on a Perspective Biosystems Mariner BiospectrometryTOF mass spectrometer.

The HPLC chromatogram shown in FIG. 2 shows a complete profile of thecompounds identified from a 3:1 ethanol:water extract of EO. The majorcompounds are mucic acid 2-gallate (7), followed by gallic acid (1)which co elutes with GG1 (4). Gallic acid (1) and GG1 (4) can beseparated by additional HPLC separation. It should be noted that themucic acid gallates derivatives 7 and 9 equilibrate in aqueous solutionsas previously reported [22], while 1-methyl mucate-2-gallate (8) is notan artifact of the isolation procedure as it could be isolated when onlyethanol, water and acetonitrile were used.

Example 2 Effect of EuMil on Cholesterol, LDL Cholesterol andTriglycerides in Rabbits

As shown in FIG. 2, rabbits fed a high fat and cholesterol diet forthree months had markedly elevated level (17-fold higher as compared tocontrol) of total plasma cholesterol (850 mg/dl). In contrast, the levelof plasma cholesterol in control rabbits, fed EuMil (1 gm/kg of rabbitchow) and EuMil plus fat was similar up to four months.

As expected, most of the increase in cholesterol in rabbits fed a highfat and cholesterol diet was associated with plasma low densitylipoproteins (FIG. 3). In contrast, the plasma LDL cholesterol level incontrol rabbits, EuMil and EuMil plus fat fed rabbits were similar.

Following fat feeding for two-four months, the plasma level oftriglycerides, shown in FIG. 4, was within the normal range. Feedingrabbits EuMil alone (throughout the four months of experimentation) didnot alter the plasma level of triglycerides significantly as compared tocontrol.

Example 3 Effect of EuMil on Cholesterol, LDL, Cholesterol Triglyceridesand Apolipoprotein Levels in Human Subjects

Next, experiments were carried out in human subjects to determine theeffect of EuMil on cholesterol, LDL, cholesterol triglycerides andapolipoprotein levels. The studies conducted in human subjects (normalmale and female), shown in FIG. 5, revealed the following. After 2 weeksof oral intake of EuMil (daily 500 mg) the levels of total cholesterol,triglycerides, LDL, VLDL decreased significantly.

The level of two pro-atherogenic apolipoproteins apolipoprotein B and Lp(a) also decreased markedly. Interestingly, the level of apoA-1, ananti-atherosclerotic apolipoprotein, which has been previouslyimplicated in cholesterol efflux, increased upon EuMil treatment.

Example 4 Effect of EuMil and its Major Herbal Ingredients on theBiosynthesis of Cholesterol

As shown in FIG. 6, lovastatin markedly decreased the biosynthesis ofcholesterol (down to ˜20% compared to control) and cholesteryl esters(˜50% compared to control). In contrast, crude aqueous extracts of O.Sanctum (JH-1 crude), EO (JH-2 crude), W. somnifera (JH-3 crude) andEuMil (JH-4 crude) marginally inhibited or did not inhibit cholesterolsynthesis in ASMC (FIG. 5). However, several of these herbssignificantly inhibited cholesteryl ester synthesis. Polyamide was usedto remove polymeric tannins from the aqueous extracts of EuMil and itsherbal ingredients and the resulting fractions were subjected tocell-based assays.

The results presented herein demonstrate that EuMil, an herbalformulation, can markedly decrease total, free and LDL cholesterol in arabbit model of hypercholesterolemia and in man via activity of the EOcomponent of EuMil. The data demonstrates that this reversal is, incertain examples, a complete reversal. The results indicate that thehypocholesterolemic activity in EuMil resides within its EO componentand lastly, the hypocholesterolemic active compounds identified in EOare derivatives of gallate ester of mucic acid (7, 8 and 9), glucose (4,5 and 6) and glycerol (3), gallic acid (1) and methyl gallate (2a) orethyl gallate (2b), depending upon the extraction procedure.

Previous studies have also shown that feeding a diet high in fat andcholesterol induces marked hyperlipidemia in rabbits. This resultspresented herein are in agreement with these studies. For example, after2 months of feeding rabbits a high fat and cholesterol diet there was amodest increase in cholesterol which increased ˜17 fold after 3 months.Most of this could be attributed to an increase in LDL cholesterol.However, the level of triglyceride remained within the normal rangethroughout the 3-4 months of experimentation in the EuMil/fat andcholesterol fed rabbits. One important observation in the present studywas that in rabbits fed EuMil hypercholesterolemia was almost entirelyabrogated, as compared to fat fed rabbits. Feeding rabbits EuMil plusrabbit chow alone did not alter plasma cholesterol and triglyceridelevels. Preliminary studies in two normal human subjects demonstratedthat the consumption of 500 mg of EuMil daily for 15 days reduced thelevels of total and lipoprotein-associated cholesterol andtriglycerides. Similarly, the level of two atherogenic lipoproteins(i.e. apolipoprotein Band Lp(a)) also were decreased. The unexpectedobservation was an increase in the level of apolipoprotein A-I.Apolipoprotein A-I is a major protein component in high densitylipoproteins and plays a critical role in “reverse cholesteroltransport” by way of facilitating the efflux of cholesterol fromperipheral tissues and transporting them back to the liver. Thesepreliminary observations suggest cholesterol efflux as a beneficialbiochemical mechanism by which EuMil may lower blood level ofcholesterol in addition to inhibition of cholesterol biosynthesis andcholesterol uptake.

Example 5 Measurement of the Effect of Mevalonate on the IntermediarySteps in Cholesterol Biosynthesis

Previous studies have shown that the mechanism by which lovastatininhibits cholesterol biosynthesis involves the inhibition of HMG-CoAreductase activity and this could be bypassed by feeding cellsmevalonate [3]. To determine if the hypocholesterolemic molecule derivedfrom EuMil also recruits the above mechanism of inhibition ofcholesterol synthesis the following experiment was conducted. Cells wereincubated with and without mevalonate (1 mM) and the incorporation of[14C] acetate into various metabolites in the biosynthetic pathwayleading to cholesterol synthesis was measured. Briefly, the radiolabeledlipids extracted from cells were dried in nitrogen and subjected tosaponification using 1.5 ml of 90% KOH and 1 ml of ethanol. Followingincubation for 2 h at 95oC the samples were extracted with diethyl etherand next, washed with 50% ethanol. The extracts were then subjected toHPTLC using benzene-ethyl acetate (1:5 by volume) as an eluent and eachplate was calibrated with squalene, lanosterol, coenzyme Q andcholesterol. Following development, the plates were dried in air andsubjected to autoradiography. Finally, the gel areas corresponding tovarious metabolites were scraped and radioactivity measured byscintillation spectrometry

Example 6 Effect of Active Compounds on Cholesterol Biosynthesis andCholesterol Mass

Next, the effect of the active compounds shown in FIG. 1 and Table 1 oncholesterol biosynthesis was examined. FIG. 8 shows that severalfractions derived from EO exhibited a dose-dependent (over a range of 5μg/ml to 0.31 μg/ml) decrease in cholesterol biosynthesis.

Next, the effect of the active compounds shown in FIG. 1 and Table 1 oncholesterol mass was examined. As shown in FIG. 9, several activefractions derived from EO extract markedly inhibited the cellular massof cholesterol in ASMC.

Example 7 Effect of Active Fractions on Cholesterol Efflux andCholesterol Uptake

Next, experiments were performed to examine if the active fractions hadan effect on cholesterol efflux and uptake. Several of active fractionssignificantly increased the efflux of cholesterol from cultured H-ASMC,as shown in FIG. 10. For example, fractions 152, 119V, 137B and 139Dmarkedly induced the efflux of cholesterol from cells. Gallic acid alsowas effective in inducing cholesterol efflux.

Lovastatin was not effective in inhibition of cholesterol uptake inH-ASMC. However, many active fractions were found to inhibit the uptakeof cholesterol, as shown in FIG. 11.

Example 8 Crude Versus Polyamide Treated Extract

Previous Ayurvedic studies have suggested that several herbal mixturesexert their effect in a synergistic manner Moreover, since EuMil is madeup of a mixture of EO, W somnifera and O. sanctum (with Asparagusracemosus used as a filler for pill preparation), our effort was focusedon whether this herbal preparation exerted its hypocholesterolemiceffect in a synergistic manner or whether the activity was confined toonly one of the above herbal ingredients. Since the crude extracts fromEuMil and the herbal preparations above contained large amounts oftanins, the extracts of EuMil and the individual plants were passedthrough polyamide to remove polymeric tannins, It was found that onlythe EO and EuMil extracts markedly inhibited cholesterol syntheses incell-based assays in H-ASMC.

Next, it was decided to look at the difference between the crude extractand the polyamide treated extract. The 25% ethanol extract (115 mg) waseluted through polyamide with 100 ml each of the following solvents:25%, 50% and I00% ethanol in water. The fractions were combined (35 mg)and analysed by HPLC which is shown in FIG. 12.

Mucic acid and its derivatives and gallic acid were not present afterthe polyamide treatment by compare the chromatograms of the crudeextract (FIG. 7) and polyamide treated extract (FIG. 12). This resultwas confirmed by MS and NM experiments. Fraction C showed a mass of m/z484 which is a derivative of compound DGG 16 (6). The rest of thefractions grouped as D, showed a common mass of m/z 332 thatcorresponded to derivatives of gallic acid attached to one sugar. Asdiscussed previously, ethyl gallate (2b) is an artifact formed duringextraction with ethanol. The cholesterol lowering activity shown by theinitial polyamide of the crude EO plant extract′ was due to the presenceof enriched amounts of gallic acid derivatives attached to glucose. Thelatter part showed peaks that correspond to higher molecular weightgallotans present in considerably lower amounts; therefore, polyamidetreatment is not an enrichment efficient step as the many of thecompounds of interest, especially the mucic acid derivatives, are lostduring the process.

The studies presented herein demonstrate that the hypocholesterlemicactivity in EuMil resides predominantly with its EO component. Theresults show that the hypocholesterolemic active compounds arederivatives of gallate ester of mucic acid (7, 8 and 9), glucose (4, 5and 6) and glycerol (3), gallc acid (1) and methyl gallate (2a) or ethylgallate (2b) depending on the extraction procedure. The studies usingcultured aortic smooth muscle cells reveal that the active compoundslower cholesterol level by a combination of biochemical mechanisms suchas inhibition of cholesterol biosynthesis, inhibition of cholesteroluptake and subsequent cholesterol efflux. The studies indicate that theactive compounds from EO may recruit multiple mechanisms, unlike thestatins, to serve as hypocholesterolemic agents.

As presented herein, the biological/functional studies using the activecompounds revealed that gallate had an effect on cell proliferation andapoptosis, while many of the other compounds did not have the sameeffect. Lovastatin was used as a positive control throughout the studiesand also did not appear to inhibit cell proliferation. Thus, most of theactive components in EO are not toxic to H-ASMC. Similar observation wasmade using human umbilical vein endothelial cells and normal humanproximal tubular cells (data not shown). In agreement with the cellbased assays shown herein that demonstrated the inhibition ofcholesterol biosynthesis by active components, the cholesterol mass inthese cells was also reduced compared to control. In fact, severalactive fractions were as good or better then lovastatin in loweringcholesterol level in H-ASMC.

The active compounds lowered cholesterol level by a combination ofbiochemical mechanisms including, but not limited to, inhibition ofcholesterol biosynthesis, inhibition of cholesterol uptake andsubsequent cholesterol efflux.

In fact, several active fractions were as good or better then lovastatinin lowering cholesterol level in human ASMC. The results describedherein show that several active compounds markedly inhibit the uptake ofcholesterol, in particular, methyl gallate (2a). DGG16 (6), GG6 (5) andmucic acid gallate derivatives 7, 8 and 9 were all effective indecreasing cholesterol level in human ASMC. On the other hand, gallatederivatives of mucic acid, methyl gallate and gallate were also veryeffective in stimulating the efflux of cholesterol from these cells.Previous extensive studies have elaborated the role of ABC-A1transporters and sterol binding and sterol receptor bindinggenes/proteins in regulating cholesterol efflux and uptake ofcholesterol [7]. It is also well known that the lipid metabolizing genessuch as apolipoprotein E (apoE), apolipoprotein B (apoB), cholesterylester transporter protein (CETP) and the LDL receptor (LDLR) aretargeted by cholesterol lowering drugs such a statins. The resultspresented herein demonstrate that the active compounds from EO recruitsmultiple mechanisms in their actions as hypocholesterolemic agents,unlike the statins. Further, the strong antioxidant effect of EO and itsrelated medicinal plant also may play a role in reducing cholesterol[54].

In a recent study it was shown that in ASMC aggregated LDL increased andsimvastatin reduced cholesteryl ester accumulation (45). We alsoobserved that in ASMC EuMil markedly reduced ox-LDL induced cholesterylester levels. The preliminary in vitro studies suggested that one of themechanisms by which EuMil reduces cholesterol level may involveinhibition of cholesterol synthesis as well as cholesteryl estersynthesis, without any significant effect on triglyceride level.

Methods and Materials Cells

Human aortic smooth muscle cells (H-ASMC) were purchased from Cambrex(Walkersville, Md,). All chemicals were purchased from Sigma ChemicalCompany (St. Louis, Mo.). [3H]Thymidine (5-10 Ci/mmol) and [14C]acetate(50-60 mCi/mmol) were purchased from American Radiolabeled Company (St.Louis, Mo.). High performance thin layer chromatography plates (silicagel 60 Å, Whatman) were purchased from Fisher Scientific (Pittsburgh,Pa.).

EuMil and Plant Material

Dry powder of EuMil herbal formulation, W. somnifera, O. sanctum and EOwere provided by Indian Herbs International, Saharanpur, India. A stocksolution of EuMil and its ingredients (1 mg/ml) were dissolved in 12.5%DMSO in water for cell-based assay and immediately used afterpreparation.

Preparation of Oxidized LDL and its Characterization

Human plasma low density lipoproteins (d 1.022-1.063 g/ml) were preparedfrom the plasma of normalipidemic controls. This LDL was subjected tooxidation with 10 μM CuSO4 and characterized as described earlier [12].

Incubation of Cells with EuMil/Ingredient Fractions

H-ASMC (×10⁵) were seeded in plastic 6-well trays and grown in smoothmuscle cells medium-2 (Cambrex). Incubation was pursued in awater-jacketed incubator with 5% CO2 and 95% air. After the cellsreached confluence medium was replaced by plain serum-free medium.Following incubation for 24 h fresh medium, 100 μg/ml ox-LDL and variousherbal fractions solubilized in 12.5% DMSO were added. Cells that wereincubated with lovastatin (10 μM) served as a positive control in allexperiments.

Rabbits and Analysis of Lipids and Lipoproteins

New Zealand white male rabbits (2 months old) were purchased from alocal supplier and housed in a sterile/germ free animal facilities for2-3 days prior to experimentation. Rabbits (5 lbs each), three in eachgroup were fed the following diet for four months. These were: a) arabbit chow; b) chow plus EuMil (1 gm/kg chow); EuMil plus fat (14%coconut oil and 0.2% cholesterol) and d) 14% coconut oil and 0.2%cholesterol. At one, two, three and four month intervals, the rabbitswere bled by venipuncture. Plasma was prepared and lipoproteins (LDL)were isolated by ultracentrifugation. The level of cholesterol,triglycerides and LDL cholesterol was measured employing standardizedlaboratory procedures at the Johns Hopkins Lipid and LipoproteinLaboratory using NCEP guidelines.

Human Subjects and Analysis of Lipids and Lipoproteins

Fasting blood from one normal male and one normal female was analyzedfor the levels of lipids and lipoproteins. They took one 500 mg pill ofEuMil daily for two weeks without any dietary restrictions. Blood wasdrawn before and after EuMil consumption for 2 weeks and lipid andlipoprotein levels were measured.

Cell-Based Assay:

The cell-based assay was used follow the activity during isolation ofthe EO fruit powder active principles. The active components were polarin nature and a C-18 HPLC column designed specifically for the isolationof polar compounds was used. The active compounds, which were isolatedusing preparative HPLC (see FIG. 9 for representative chromatogram),were gallic acid (1), methyl gallate (2a)/ethyl gallate (2b) dependingon the extraction solvent used, glycerol-1-gallate (3),glucose-1-gallate (GG1) (4), glucose-6-gallate (GG6) (5),glucose-1,6-digallate (DGG16) (6), mucic acid-2-gallate (7), methylmucic acid-2-gallate (8) and mucic acid 1,4-lactone 5-gallate (9).

Measurement of Cell Proliferation

Following incubation for 18 h with and without ox-LDL andEuMil/ingredients and lovastatin [3H]thymidine (5 μCi/ml) was added in96 well dishes and incubation continued for 6 h at 37oC. Next, the cellswere washed and the incorporation of [3H]thymidine into DNA was measuredemploying scintillation spectrometry [12]. The data was calculated asdpm/well. All experimental values were expressed as a percentage of thecontrol values (cells incubated with ox-LDL alone).

Measurement of Apoptosis

ASMC (×10⁴) were seeded on sterile glass cover slips and incubated inthe presence/absence of active fractions of EO, lovastatin, ox-LDL for24 h. Cells were fixed with ethanol-acetic acid (3:1 by volume) at roomtemperature for 10 min, washed three times with phosphate bufferedsaline (pH 7.4) and stored at −20oC. Next cell were stained with DAPI(4′,6-diamidino-2-phenylindole dihydrochloride) reagent and nuclei werevisualized by fluorescence microscopy (Zeiss Axiovert 25).

Measurement of Cholesterol Efflux

For cholesterol efflux studies near confluent cells were labeled with[3H]-cholesterol for 24 h, washed, and incubated for 24 h with theherbal fractions in serum-free DMEM medium. Percentage efflux wascalculated by subtracting the radioactive counts in the blank media fromthe radioactive counts in the presence of an acceptor, and then dividingthe result by the sum of the radioactive counts in the medium plus thecell fraction.

Determination of Cholesterol Uptake

Confluent culture of human aortic smooth muscle cells were pre-incubatedin serum-free DMEM for 24 h. Next, fresh medium and [3H]cholesterol (5μCi/ml) plus various herbal compounds were added. Following incubationfor 24 h and 48 h, [3H]cholesterol taken up by cell was measured bysolubilization in 1 M NaOH and radioactivity measured.

Measurement of the Level of Cholesterol

Following treatment cells were extracted with hexane-isopropanol 3:2 byvolume as described above. The pooled extracts were dried in a stream ofnitrogen and subjected to measurement of total cholesterol mass using acholesterol kit (WAKO chemicals, VA) with cholesterol as the standard.The data was expressed as μg cholesterol per mg protein.

Measurement of Cholesterol Biosynthesis in Cultured Cells

Cells were incubated with and without ox-LDL and EuMil/ingredients and[14C] acetate (1 μCi/ml, 24 h). Next, medium was removed; the monolayerswere washed three times with PBS. Cells were extracted with hexane:isopropanol (3:2 by volume, 2×3 ml) at room temperature. The extractswere pooled in glass tubes and total lipid extract dried in nitrogenatmosphere. 1N NaOH (1 ml) was added to each tube to solubilize proteinsand the incubated overnight in open air. Next, water (2 ml) was added tothe air dried tubes, the contents were mixed and utilized for proteinmeasurement using BCA protein assay kit (Pierce, Rockford, Ill.)

The total lipid extracts were dried in stream of nitrogen, re-suspendedin chloroform-methanol (2:1 by volume) and applied on a HPTLC silica gelplate. Standard solutions of cholesterol, monolein, diolein, triolein,fatty acid and cholesteryl oleate (50 _(l)ig each) also were applied tothe plate. The plates were developed in heptane-ether-acetic acid(85:15:1 by volume). Following development, the plates were dried in airand exposed to an X-ray film for 2-3 days at −800C. After the films weredeveloped, the HPTLC plates were immersed in iodine vapors to identifythe migration of individual neutral lipid molecular species. Gel areas,corresponding to cholesterol were scraped and radioactivity was measuredusing Beckman liquid scintillation spectrometer. The data was expressedas counts per mg protein. The incorporation of radioactivity in cellsincubated with oxidized LDL was considered as 100% (control). Allexperimental values in cells treated with EuMil/ingredients plusoxidized LDL were expressed as a percentage of the control. All assayswere conducted in triplicate.

The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements of this invention and stillbe within the scope and spirit of this invention as set forth in thefollowing claims.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

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1.-47. (canceled)
 48. A pharmaceutical composition for the treatment orprevention of an elevated blood lipid level-related disease or disordercomprising one or more gallic acid derivatives and a pharmaceuticallyacceptable excipient.
 49. The pharmaceutical composition of claim 48,wherein the gallic acid derivative is derived from EuMil.
 50. Thepharmaceutical composition of claim 48, wherein the one or more gallicacid derivatives is selected from the group comprising: methyl gallate,ethyl gallate, glycerol-1-gallate, glucose-1-gallate (GG1),glucose-6-gallate (GG6), glucose-1,6-digallate (DGG16), mucicacid-2-gallate, 1-methyl mucate-2-gallate, mucic acid 1,4-lactone5-gallate, geraniin, corilagin, chebilc acid, and m-digallic acid withminor p-digallic acid.
 51. The pharmaceutical composition of claim 48,comprising a combination of gallic acid derivatives selected from thegroup consisting of: Compound 4+Compound 5+Compound 2a, Compound4+Compound 8+Compound 2a, Compound 4+Compound 5+Compound 7, Compound4+Compound 2a, Compound 4+Compound 2b, Compound 7+Compound 2b, Compound5+Compound 8, and Compound 5+Compound
 7. 52. The pharmaceuticalcomposition of claim 48, wherein the at least one gallic acid derivativeis present in an amount from about 10 mg-500 mg.
 53. The pharmaceuticalcomposition of claim 52, wherein the at least one gallic acid derivativeis present in an amount from about 40 mg-200 mg.
 54. The pharmaceuticalcomposition of claim 48, further comprising a one or more second agents.55. The pharmaceutical composition of claim 54, wherein the one or moresecond agents is a therapeutic agent.
 56. The pharmaceutical compositionof claim 55, wherein the therapeutic agent is selected from the groupconsisting of: inhibitors of cholesterol metabolism, inhibitors oftriglyceride synthesis, beta blockers, diuretics, inhibitors of plateletaggregation, angiogenesis inhibitors, arthritis medication, toxins,anti-inflammatory agents.
 57. The pharmaceutical composition of claim55, wherein the therapeutic agent is attached to the gallic acidderivative by a covalent linkage.
 58. The pharmaceutical composition ofclaim 48, wherein the composition is administered in a dosage formselected from the group consisting of a: food composition, tablet, pill,gel, capsule, suspension tablet, liquid, solution, patch, aqueousemulsion powder, lozenge, sachet, cachet, elixir, suspension, emulsion,solution, syrup, aerosol, ointment, soft gelatin capsule, and hardgelatin capsule, suppository, creams, lotions, solutions, gels, pastes,powder, and suspension.
 59. The pharmaceutical composition of claim 58,wherein the food composition is a health food product, a food productmade from cereal flour, gums, a dairy product, a soup, a broth, a paste,a sauce, a beverage, a powder, a vitamin complex, a food rich incholesterol, salt, or pepper.
 60. The pharmaceutical composition ofclaim 58, wherein the dosage form is further selected from the groupconsisting of immediate release, sustained release, and delayed release.61. The pharmaceutical composition of claim 58, wherein the dosagecomprises about 0.1% to about 95% gallic acid derivative weight toweight of the composition.
 62. A kit comprising a pharmaceuticalcomposition according to claim 48, and instructions for use.
 63. A kitcomprising one or more gallic acid derivatives and instructions for use.64. The kit of claim 63, wherein the one or more gallic acid derivativesis selected from the group comprising: methyl gallate, ethyl gallate,glycerol-1-gallate, glucose-1-gallate (GG1), glucose-6-gallate (GG6),glucose-1,6-digallate (DGG16), mucic acid-2-gallate, 1-methylmucate-2-gallate, mucic acid 1,4-lactone 5-gallate, geraniin, corilagin,chebilc acid, and m-digallic acid with minor p-digallic acid, andinstructions for use.
 65. The kit of claim 64, comprising a combinationof gallic acid derivatives selected from the group consisting of:Compound 4+Compound 5+Compound 2a, Compound 4+Compound 8+Compound 2a,Compound 4+Compound 5+Compound 7, Compound 4+Compound 2a, Compound4+Compound 2b, Compound 7+Compound 2b, Compound 5+Compound 8, andCompound 5+Compound
 7. 66. The kit of claim 63, wherein said kit is foruse in preventing or treating an elevated blood lipid level relateddisease or disorder in a subject, for use in preventing or treatinginflammation in a subject, for use in preventing or treating a stressresponse in a subject, for use in reducing cholesterol biosynthesis in asubject, for use in increasing the cellular efflux of cholesterol in asubject, or for use in inhibiting the cellular uptake of cholesterol ina subject.
 67. The kit of claim 63, wherein the one or more gallic acidderivatives is administered as a nutraceutical.